Tiny architects: biogenesis of intracellular replicative niches by bacterial pathogens

Tiny architects: biogenesis of intracellular replicative niches by bacterial pathogens Abstract Co-evolution of bacterial pathogens with their hosts led to the emergence of a stunning variety of strategies aiming at the evasion of host defences, colonisation of host cells and tissues and, ultimately, the establishment of a successful infection. Pathogenic bacteria are typically classified as extracellular and intracellular; however, intracellular lifestyle comes in many different flavours: some microbes rapidly escape to the cytosol whereas other microbes remain within vacuolar compartments and harness membrane trafficking pathways to generate their host-derived, pathogen-specific replicative niche. Here we review the current knowledge on a variety of vacuolar lifestyles, the effector proteins used by bacteria as tools to take control of the host cell and the main membrane trafficking signalling pathways targeted by vacuolar pathogens as source of membranes and nutrients. Finally, we will also discuss how host cells have developed countermeasures to sense the biogenesis of the aberrant organelles harbouring bacteria. Understanding the dialogue between bacterial and eukaryotic proteins is the key to unravel the molecular mechanisms of infection and in turn, this may lead to the identification of new targets for the development of new antimicrobials. host/pathogen interactions, vacuolar bacterial pathogens, vacuole biogenesis, membrane traffic HUNTER GATHERERS VERSUS SETTLERS AND THE INBETWEENERS: INTRACELLULAR LIFESTYLES OF BACTERIAL PATHOGENS The development and establishment of strategies allowing bacteria to survive and proliferate inside eukaryotic cells traces back in ancient times and has shaped the evolution of eukaryotic cells as we know them. Bacteria use eukaryotic cells as safe houses, providing sanctuary from a harsh environment, to access nutrients as well as means of transportation. Harbouring bacteria can be either beneficial, neutral or detrimental for eukaryotic cells (Steinert, Hentschel and Hacker 2000). The endosymbiotic theory represents the best example of how interactions between prokaryotes and eukaryotes can be beneficial, to the point where bacteria evolve to become respiratory organelles: mitochondria for animal cells (likely derived from alphaproteobacteria ancestors) (Thrash et al.2011) and chloroplasts for plant cells (possibly related to cyanobacteria) (Falcón, Magallón and Castillo 2010). On the opposite side of the scale, infectious diseases stand as a threatening example of how interactions between bacteria and eukaryotic cells can be deleterious for the latter. The first step towards the establishment of an intracellular lifestyle is the internalisation within a eukaryotic cell. Access to phagocytic cells is passively achieved due to the clearance activity of macrophages. Internalisation within non-phagocytic cells on the other hand is mediated either by bacterial surface proteins, which activate receptor-mediated endocytosis signalling pathways, or by bacterial effector proteins, which are injected into the host cell cytosol to drive the reorganisation of the host cell cytoskeleton and bacterial engulfment. These two processes are commonly referred to as zippering and triggering mechanisms, respectively (Cossart 2004). Despite the numerous advantages associated with an intracellular lifestyle, the cellular environment remains inhospitable for bacteria: inside endosomes, nutrients are scarce and material internalised either by endocytosis or phagocytosis is destined to lysosomal degradation; on the other hand, the cytosol is rich in nutrients, but constantly surveyed by the innate immune system. Bacteria have adapted to this environment by adopting very different lifestyles, reminiscent of those observed in early humans: cytosolic bacteria escape the degradative pathway by rupturing internalisation vacuoles thus accessing the cytosol. Here, bacteria including Listeria monocytogenes, Shigella flexneri, Rickettsia species and Mycobacterium marinum and Burkholderia live like hunters-gatherers, harnessing the actin cytoskeleton to escape the autophagy-mediated innate immune surveillance, gather nutrients and explore the intracellular environment to access bystander cells (Gouin, Welch and Cossart 2005) (Fig. 1). Of note, intracellular motility can also be fuelled by flagellar motors (Knodler et al.2010) or by a combination of the two (French et al.2011). On the other hand, vacuolar bacteria settle within internalisation vacuoles and re-shape cellular compartments and organelles to create new environments, which are derived from eukaryotic components but only exist in the context of bacterial infections (Cossart and Roy 2010) (Fig. 1). As always in nature, this dichotomy is not 100% accurate. Thus, given the right environment or conditions, some cytosolic bacteria can also replicate within membrane-bound compartments and conversely, an increasing number of vacuolar pathogens escape their niche to replicate in the cytosol (Fig. 1). This review will mainly focus on vacuolar bacteria and the many strategies developed to generate pathogen-specific replicative niches exploiting the resources of host cells. Figure 1. View largeDownload slide Intracellular lifestyles of bacterial pathogens. Upon internalisation within host cells, bacteria of the genus Chlamydia, Legionella, Ehrlichia, Anaplasma, Brucella and Coxiella remain confined within their internalisation vacuole, which they modify by driving pathogen-specific interactions with membrane-bound organelles and/or transport carriers to develop a replicative niche. Depending on their adaptation to an acidic environment, vacuolar pathogens can escape the endosomal maturation pathway by acquiring markers of organelles such as the endoplasmic reticulum (Legionella and Brucella) or the Golgi complex (Chlamydia and Brucella). Other microbes including Listeria, Rickettsia, Shigella and Burkholderia species as well as Mycobacterium marinum escape the internalisation vacuole and replicate in the cytoplasm where they can use actin- or flagellar-based motility to escape the innate immune surveillance and infect bystander cells. Finally, Mycobacterium, Salmonella, Francisella and Listeria may adopt a dual lifestyle, which includes both vacuolar and cytoplasmic stages. Inc, inclusion; LCV, Legionella-containing vacuole; BCV, Brucella-containing vacuole; CCV, Coxiella-containing vacuole; MCV, Mycobacterium-containing vacuole; SCV, Salmonella-containing vacuole; FCV, Francisella-containing vacuole; LisCV, Listeria-containing vacuole. Figure 1. View largeDownload slide Intracellular lifestyles of bacterial pathogens. Upon internalisation within host cells, bacteria of the genus Chlamydia, Legionella, Ehrlichia, Anaplasma, Brucella and Coxiella remain confined within their internalisation vacuole, which they modify by driving pathogen-specific interactions with membrane-bound organelles and/or transport carriers to develop a replicative niche. Depending on their adaptation to an acidic environment, vacuolar pathogens can escape the endosomal maturation pathway by acquiring markers of organelles such as the endoplasmic reticulum (Legionella and Brucella) or the Golgi complex (Chlamydia and Brucella). Other microbes including Listeria, Rickettsia, Shigella and Burkholderia species as well as Mycobacterium marinum escape the internalisation vacuole and replicate in the cytoplasm where they can use actin- or flagellar-based motility to escape the innate immune surveillance and infect bystander cells. Finally, Mycobacterium, Salmonella, Francisella and Listeria may adopt a dual lifestyle, which includes both vacuolar and cytoplasmic stages. Inc, inclusion; LCV, Legionella-containing vacuole; BCV, Brucella-containing vacuole; CCV, Coxiella-containing vacuole; MCV, Mycobacterium-containing vacuole; SCV, Salmonella-containing vacuole; FCV, Francisella-containing vacuole; LisCV, Listeria-containing vacuole. TOOLS FOR THE JOB: SECRETION SYSTEMS AND EFFECTOR PROTEINS To establish a communication with their recipient host cell and manipulate their machineries, pathogenic Gram-negative intracellular bacteria developed several secretion systems, which are complex macromolecular nanomachines that span both their inner and outer membranes to permit the translocation of effector proteins from the bacterial cytosol to the host cell cytosol. Among the nine existing secretion system, only type 3, 4 and 6 (T3SS, T4SS and T6SS, respectively) are capable of protein translocation (Costa et al.2015). Each intracellular bacterial pathogen needs to shape its own replicative niche. To do so, these bacteria use type 3 (Salmonella, Chlamydia, Simkania) or type 4 (Brucella, Legionella, Coxiella, Anaplasma, Ehrlichia) secretion systems. Genes encoding effector proteins often possess specific promoter sequences that are co-regulated with genes encoding their respective secretion system. Furthermore, effector proteins often carry N-terminal or C-terminal secretion signals (features including positive charges, basicity, hydrophobicity and secondary structures); eukaryotic localisation signals, which target the bacterial protein to specific host cell compartments; eukaryotic-like domains, which are often involved in the manipulation of host cell functions; and other functional domains (Escoll et al.2016). Thus, bacterial effector proteins can either mimic or modify endogenous proteins to alter host cell pathways (Dean 2011; Pearson et al.2015). The expression and secretion of effector proteins can be triggered by external signals. For example, Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system (T3SS) gene expression is activated by acidic pH, decrease in inorganic phosphate, magnesium and calcium concentrations (Löber et al.2006). In macrophages, this leads to an upregulation of SPI-2-dependent effector genes and a downregulation of the Salmonella pathogenicity island 1 (SPI-1) T3SS-dependent effector genes (Srikumar et al.2015). For Coxiella and Brucella, acidification of their respective bacteria-containing phagosomes activates their metabolism which then triggers T4SS activation and effector secretion (Boschiroli et al.2002; Newton, McDonough and Roy 2013). In the case of Legionella pneumophila, a subset of effector proteins are ‘pre-synthesised’ at later stages of the intracellular cycle, prior to host cell lysis, and translocation is triggered upon contact between the Dot/Icm T4SS and the plasma membrane of bystander cells (Charpentier et al.2009). Given their importance in the infectious cycle and virulence of intracellular bacterial pathogens, the identification and characterisation of effector proteins has fostered extensive research. Bioinformatics analysis of bacterial genomes carrying T3SS (Arnold et al.2009; Tay et al.2010; Hobbs et al.2016) and T4SS (Bi et al.2013; Lifshitz et al.2013; Meyer et al.2013; Wang et al.2014) was used to identify ‘eukaryotic-like’ genes (EUGENs) encoding hypothetical proteins with eukaryotic-like domains and motifs: ankyrin repeats, SEL1 (TPR), Set domain, Sec7, serine threonine kinase domains, U-box, F-box, Src homology 2, pentatricopeptide repeat domains, phosphorylation motifs targeted by host kinases and prenylation domains (Dean 2011; Gomez-Valero et al.2011). Results from these studies were corroborated by experimental data arising from comprehensive genetic screens using adenylate cyclase and beta lactamase fusion assays (Chen et al.2010; Carey et al.2011; Zhu et al.2011). The overall results of bioinformatics and experimental analysis were used in machine learning approaches to further characterise potential effector proteins (Lifshitz et al.2014; Martínez-García, Ramos and Rodríguez-Palenzuela 2015; An et al.2017). First identified in Legionella and Chlamydia (Cazalet et al.2010), EUGENs share a high degree of similarity with eukaryotic proteins, suggesting an initial acquisition, via horizontal gene transfer, from eukaryotic organisms (Lurie-Weinberger et al.2010; Gomez-Valero and Buchrieser 2013). Conversely, other effectors may have arisen from convergent evolution. These carry functional domains that are capable of interacting with and/or modifying eukaryotic targets and that possess no sequence homology with known eukaryotic domains or proteins. Finally, some bacterial effectors combine these two types of domains. Legionella effector SidM/DrrA appears as a prime example of such rearrangement. It possesses three domains: an N-terminal adenosine monophosphate transferase (AMPylase) domain, a central guanosine exchange factor (GEF) domain and a C-terminal phosphatidylinositol-4-phosphate binding domain (P4M). While the AMPylase domain shares structural similarities and an important catalytic motif with known eukaryotic glutamine synthase adenylyl transferases (Müller et al.2010), the GEF and P4M domains are structurally distinct from any know eukaryotic domain with similar functions (Schoebel et al.2010; Suh et al.2010). It is remarkable to observe how translocated effector proteins are unique from one species of intracellular bacteria to another and specifically manipulate host cell mechanisms and pathways to define and expand the adequate niche to support bacterial replication. Salmonella enterica serovars cause a range of diseases in humans from gastroenteritis to typhoid fever. These bacteria possess two T3SS: SPI-1, which is used for triggering internalisation of the bacteria, and SPI-2, which is essential for the biogenesis of Salmonella-containing vacuoles (SCVs). SPI-2 is used to inject 28 effectors in the host cell endomembrane system and cytosol (Figueira and Holden 2012). A remarkable feature of SCVs is the presence of Salmonella-induced tubules that radiate from their surface (Liss and Hensel 2015). Eight effector proteins tightly regulate membrane dynamics of SCVs: SifA, PipB2, SseJ, SopD2, SseF, SseG, SteA and SpvB (Knuff and Finlay 2017). While SifA and PipB2 promote the formation of Salmonella-induced filaments (SIFs), maintain SCVs membrane integrity and enable the continuous fusion of host vesicles to SCV membranes, their action is counteracted by the activities of SseJ, SopD2 and SpvB. Furthermore, SifA forms a complex with SKIP and Rab9, thus inhibiting the Rab9-dependent transport of Mannose 6-phosphate receptor (M6PR) to SCVs (McGourty et al.2012). This decreases the delivery of lysosomal enzymes to SCVs and protects intracellular Salmonella from host defences. Other effector proteins that might participate to SCV biogenesis are PipB, an SCV-interacting effector with no yet defined function (Knodler et al.2003), and GtgE, a protease that cleaves Rab29 and Rab32 off SCVs (Spano and Galan 2012). Chlamydia species are obligate intracellular bacterial pathogens that replicate in a specialised membrane compartment called the inclusion. These bacteria use a T3SS to inject between 36 and 107 effector proteins in the host cytosol, depending on the species (Elwell, Mirrashidi and Engel 2016). Chlamydia trachomatis injects CT229, which binds to Rab4 (Rzomp, Moorhead and Scidmore 2006; Ronzone and Paumet 2013), IncA (CT119), InaC (CT813) and IPAM (CT223) which act as inhibitory SNAREs to limit fusion of the inclusion with VAMP3, VAMP7 and VAMP8-positive compartments (Ronzone and Paumet 2013; Elwell, Mirrashidi and Engel 2016), and IncE (CT116) which binds Sorting Nexins 5, 6 and 32 (SNX5, SNX6 and SNX32 respectively) to disrupt host trafficking pathways and promote bacterial growth (Paul et al.2017). Finally, IncD and IncV effector proteins localise at endoplasmic reticulum (ER)-inclusion membrane contact sites (MCS) and interact with ER-resident proteins CERT, VAPA and VAPB to mediate lipid transfer and inclusion biogenesis (Derré, Swiss and Agaisse 2011). Brucella species cause brucellosis in a wide variety of domestic and wild animals, humans being secondary or accidental hosts. The bacteria uses a T4SS to secrete approximately 15 effector proteins during the course of infection, some of which perturb ER secretion (Myeni et al.2013; Ke et al.2015). Effector protein RicA is capable of interacting with the inactive form of the small Rab GTPase Rab2 and modulates the maturation of Brucella-containing vacuoles (BCVs) (de Barsy et al.2011). Another effector protein favouring the biogenesis of BCVs is SepA (Döhmer et al.2014). While the function of the protein has not been established in detail, its secretion during the early stages of infection abolishes the fusion of lysosomes with BCVs, thus favouring the establishment of a suitable replicative niche for the bacterium. Furthermore, it has been recently shown that BspB is capable of interacting with the conserved oligomeric Golgi (COG) tethering complex to redirect Golgi-derived vesicles to the BCV (Miller et al.2017). Coxiella burnetii, the causative agent of Q fever, translocates approximately 133 effector proteins (Moffatt, Newton and Newton 2015; Larson et al.2016; Qiu and Luo 2017). Bacteria develop a unique spacious, acidified vacuole (the Coxiella-containing vacuole, CCV) containing active hydrolases. Five C. burnetii effector proteins have been shown to localise at CCVs and have thus been referred to as Coxiella vacuolar proteins (CvpA to E) and mutations in cvp genes severely affect CCVs biogenesis and C. burnetii replication (Larson et al.2015). Despite the fact that their mode of action remains largely elusive, it has been reported that CvpA binds AP2 adaptor complexes on recycling endosomes, re-routing these vesicles to the CCV (Larson et al.2013) and CvpB inhibits the activity of the PI 3-kinase PIKfyve to maintain vacuolar phosphatidylinositol 3-phosphate (PI(3)P), which is used for homotypic vacuole fusion via the autophagy machinery (Newton et al.2014; Kohler et al.2016; Martinez et al.2016). In addition, Cig57 interacts with components of the clathrin-mediated vesicular trafficking and is required for vacuole biogenesis (Latomanski et al.2016) and CirA recruits and stimulates the GTPase activity of RhoA at the CCV (Weber et al.2016b). A Coxiella evolutionary related pathogen is L. pneumophila, the causative agent of Legionnaire's disease. This bacterium also possesses a T4SS whose structure has recently been characterised (Ghosal et al.2017). At least 330 effectors have been identified for this pathogen and approximately 40 of these have been linked to the manipulation of vesicle identity and trafficking to favour the biogenesis of Legionella-containing vacuoles (LCVs) (Qiu and Luo 2017). At least six effector proteins play a role in the recruitment, stabilisation and recycling of the small GTPase Rab1 on LCVs: SidM/DrrA, AnkX, LepB, SidD, Lem3 and LidA (Qiu and Luo 2017). Additionally, the effector protein RalF functions as an ARF1 GEF on LCVs (Nagai et al.2002). Activated ARF1 may promote recycling of coat proteins from the LCV back to the ER, thus favouring the expansion of the LCV (Nagai et al.2002). Other effector proteins participate in the biogenesis of LCVs include VipD, a Rab5-interacting effector with a phospholipase A1 activity that inactivates PI(3)P on early endosomes, thus changing their lipid composition and inhibiting their fusion with LCVs (Gaspar and Machner 2014). The effector protein SidK blocks the acidification of LCVs by inhibiting the activity of the vacuolar ATPase (v-ATPase) that pumps protons across membranes (Xu et al.2010). Finally, RavZ is a secreted cysteine protease that inhibits host autophagy by selectively deconjugating LC3 from host membranes (Choy et al.2012). Mycobacterium tuberculosis is a facultative intracellular pathogen responsible for millions of casualties each year. Following internalisation, the bacterium secretes two effector proteins that are capable of arresting the maturation of Mycobacterium-containing vacuoles (MCVs). Mycobacterium secretes the acid phosphatase SapM, which has strong affinity for PI(3)P and removes this signalling lipid from MCVs, thus blocking the phagosomal maturation during infection (Saleh and Belisle 2000). Ndk is a nucleoside diphosphate kinase that inactivates the GTPases Rac1, Rab5 and Rab7. Thus, Ndk not only triggers a decrease in lysosomal fusion with bacteria-containing phagosomes but also a decrease in the production of reactive oxygen species that would be deleterious to Mycobacterium replication (Sun et al.2010, 2013). Table 1 provides a complete list of the above-mentioned effector proteins, their eukaryotic targets and function in the regulation of membrane traffic and vacuole biogenesis. Table 1. Bacterial effector proteins participating in vesicle trafficking and vacuole biogenesis, their eukaryotic targets and main function. Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) View Large Table 1. Bacterial effector proteins participating in vesicle trafficking and vacuole biogenesis, their eukaryotic targets and main function. Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) View Large LOCATION, LOCATION, LOCATION: WHERE TO BUILD A COMFORTABLE HOME As mentioned above, a vacuolar lifestyle protects bacteria from the host innate immune cytosolic surveillance. On the other hand, material internalised within endosomes and vacuoles is destined for degradation along the endocytic pathway, and the cytosol is certainly more abundant in nutrients as compared to membrane-bound compartments. Thus, a successful vacuolar lifestyle requires the evolution of strategies to deviate bacteria-containing vacuoles from the lysosomal route and to converge nutrients to the newly established replicative niche. Escaping degradation is achieved by two main strategies: either by arresting vacuole maturation at several stages between endocytic vesicles and lysosomes (Via et al.1997; Beron et al.2002; Mukherjee et al.2002; Rikihisa 2017) or by contacting host cell organelles such as the ER and the Golgi apparatus to generate unique, hybrid compartments (Hackstadt et al.1996; Tilney et al.2001) (Fig. 2). It is remarkable to note that either strategy requires extensive reprogramming of eukaryotic cell membrane trafficking and signalling pathways, which is coordinated by spatially isolated pathogens through vacuolar membranes. Vacuolar pathogens replicating within arrested endocytic vacuoles can either block endocytic maturation very early, therefore generating replicative niches with neutral pH (this is the case for M. tuberculosis and Ehrlichia chaffeensis for example), or delay their manipulation of membrane trafficking until later time points of infection, exposing themselves to acidic environments (a strategy adopted by S. enterica, C. burnetii and Brucella) (Fig. 2). Microbes belonging to the second group have adapted to acidic (and degradative in the case of C. burnetii) environments to the point that metabolism and/or translocation of effector proteins require the acidification of the replicative niche (Fig. 2). Figure 2. View largeDownload slide ‘Membrane code’ of bacteria-containing vacuoles. Early translocation of effector proteins allows L. pneumophila to drive the escape from the endocytic maturation and generate LCVs with neutral pH. This is achieved by manipulating the lipid signature of LCVs, which become mainly enriched in PI and PI(4)P, and by intersecting the early secretory pathway at ER exit sites (ERES). Thus, LCVs are decorated by ER membranes and are positive for typical secretory Rab GTPases such as Rab1, 14 and 8 (blue circles). The recruitment of a large number of Rab GTPases regulating different membrane trafficking pathways at Chlamydia inclusions is indicative of the complex interactions that this bacterium establishes with the infected cell. Transport to the MTOC facilitates interactions with the Golgi apparatus which is exploited as a source of membranes and lipids by Chlamydia. Mycobacterium uses translocated effector proteins to deplete PI(3)P from MCVs thereby perturbing the recruitment of Rab GTPases involved in membrane remodelling along the endocytic maturation pathway. As a result, MCVs are characterised by neutral pH and early endocytic (EE) Rab GTPases. Membrane damage by the bacterial secretion system may lead to membrane rupture and cytoplasmic escape of Mycobacteria. Other vacuolar pathogens require acidification of their replicative environment to activate metabolism and/or translocation of effector proteins. Salmonella SPI-2 effectors are translocated upon interactions of SCVs with late endosomal (LE) compartments and these are involved in the detoxification of SCVs to allow bacterial replication. SCVs are thus positive for early and late endocytic Rab GTPases including Rab5, 7, 11 and 14. Rab2 is also observed at SCVs and is indicative of fusion events between SCVs and COPII-positive secretory vesicles which facilitate SCVs rupture and cytoplasmic dissemination of Salmonella. Salmonella Typhimurium prevents Rab32 recruitment at SCVs, which has been demonstrated to regulate the species specificity of this pathogen. Coxiella burnetii metabolism and effector protein translocation is activated upon fusion of CCVs with lysosomes (Ly). Effector proteins co-opt the autophagy (Au) pathway, which is involved in homotypic fusion events between CCVs. Autophagy regulation is mediated by the manipulation of PI metabolism at the surface of CCVs which become enriched in PI(3)P. Thus, CCVs are positive for early and late Rab GTPases (Rab5 and 7, respectively) and also in the autophagy-related Rab24. Early Brucella-containing vacuoles (eBCVs) follow the endocytic maturation pathway until acidification of vacuolar pH upon fusion with lysosomes and the acquisition of the late endosomal Rab7. This triggers the translocation of Brucella effectors, which mediate the detoxification of BCVs by intersecting the early secretory pathway at ERES and COG complexes. Contacts with ER membranes and the acquisition of the ER-associated Rab2 drive the transformation into replicative BCVs (rBCVs) that support bacterial replication. Later during infection, rBCVs intersect the autophagy pathway and transform into autophagic BCVs (aBCVs), thus facilitating release from infected cells into the extracellular environment. Figure 2. View largeDownload slide ‘Membrane code’ of bacteria-containing vacuoles. Early translocation of effector proteins allows L. pneumophila to drive the escape from the endocytic maturation and generate LCVs with neutral pH. This is achieved by manipulating the lipid signature of LCVs, which become mainly enriched in PI and PI(4)P, and by intersecting the early secretory pathway at ER exit sites (ERES). Thus, LCVs are decorated by ER membranes and are positive for typical secretory Rab GTPases such as Rab1, 14 and 8 (blue circles). The recruitment of a large number of Rab GTPases regulating different membrane trafficking pathways at Chlamydia inclusions is indicative of the complex interactions that this bacterium establishes with the infected cell. Transport to the MTOC facilitates interactions with the Golgi apparatus which is exploited as a source of membranes and lipids by Chlamydia. Mycobacterium uses translocated effector proteins to deplete PI(3)P from MCVs thereby perturbing the recruitment of Rab GTPases involved in membrane remodelling along the endocytic maturation pathway. As a result, MCVs are characterised by neutral pH and early endocytic (EE) Rab GTPases. Membrane damage by the bacterial secretion system may lead to membrane rupture and cytoplasmic escape of Mycobacteria. Other vacuolar pathogens require acidification of their replicative environment to activate metabolism and/or translocation of effector proteins. Salmonella SPI-2 effectors are translocated upon interactions of SCVs with late endosomal (LE) compartments and these are involved in the detoxification of SCVs to allow bacterial replication. SCVs are thus positive for early and late endocytic Rab GTPases including Rab5, 7, 11 and 14. Rab2 is also observed at SCVs and is indicative of fusion events between SCVs and COPII-positive secretory vesicles which facilitate SCVs rupture and cytoplasmic dissemination of Salmonella. Salmonella Typhimurium prevents Rab32 recruitment at SCVs, which has been demonstrated to regulate the species specificity of this pathogen. Coxiella burnetii metabolism and effector protein translocation is activated upon fusion of CCVs with lysosomes (Ly). Effector proteins co-opt the autophagy (Au) pathway, which is involved in homotypic fusion events between CCVs. Autophagy regulation is mediated by the manipulation of PI metabolism at the surface of CCVs which become enriched in PI(3)P. Thus, CCVs are positive for early and late Rab GTPases (Rab5 and 7, respectively) and also in the autophagy-related Rab24. Early Brucella-containing vacuoles (eBCVs) follow the endocytic maturation pathway until acidification of vacuolar pH upon fusion with lysosomes and the acquisition of the late endosomal Rab7. This triggers the translocation of Brucella effectors, which mediate the detoxification of BCVs by intersecting the early secretory pathway at ERES and COG complexes. Contacts with ER membranes and the acquisition of the ER-associated Rab2 drive the transformation into replicative BCVs (rBCVs) that support bacterial replication. Later during infection, rBCVs intersect the autophagy pathway and transform into autophagic BCVs (aBCVs), thus facilitating release from infected cells into the extracellular environment. A remarkable example of how bacteria sense environmental pH to trigger virulence is provided by S. enterica. Upon invasion of host cells, SCVs follow the endocytic maturation pathway until merging with lysosomes (Mukherjee et al.2002). The acidification of SCVs is required for the assembly of the SPI-2 T3SS, but it is only upon insertion into the SCV membrane and sensing of the neutral pH of the host cell cytosol that effector translocation occurs (Yu et al.2010). Indeed, translocation is triggered by the dissociation and degradation of the T3SS core proteins SsaM, SpiC and SsaL, upon sensing of the cytosol neutral pH (Yu et al.2010). In order to prevent bacterial degradation by host cells, S. enterica uses SPI-2 effector proteins including SopD2 and SifA to detoxify lysosomes by subverting the activity of the small GTPases Rab7 and Rab9 (see below). Of note, recent proteomic analysis of isolated SCVs generated after 30 min and 3 h of infection highlighted an enrichment in ER-associated proteins, indicating that SCVs may also intercept the ER at nascent and intermediate stages of SCV formation (Santos et al.2015). It has been proposed that the observed membrane interactions between SCVs and the ER may provide an alternative SCV maturation pathway which resolves in the previously described vacuolar escape and cytosolic hyper-replication of Salmonella (Knodler et al.2010; Santos et al.2015) (Fig. 2). Coxiella burnetii offers another interesting example of how bacteria can adapt to harsh intracellular compartments. This microbe replicates within an acidic, degradative compartment with lysosomal features (Larson et al.2016). Differently from other pathogens following the endocytic pathway, which replicate in multiple, relatively small tight-fitting compartments, C. burnetii replicative vacuoles are extremely large and fusogenic, which leads to the generation of a single parasitophorous vacuole per infected cell (Larson et al.2016) (Fig. 2). Homotypic fusion of CCVs is facilitated by the autophagy-related SNARE protein syntaxin-17 and the C. burnetii effector protein CvpB/Cig2 (McDonough et al.2012; Newton et al.2014; Martinez et al.2016). CvpB-mediated manipulation of phosphoinositide (PI) metabolism illustrated below is likely at the basis of a subversion of the autophagy machinery to favour CCV biogenesis (Kohler et al.2016; Martinez et al.2016) (Figs 2 and 3). Emerging evidence indicates that the CCV intercept multiple membrane trafficking pathways as a mean to reroute membrane components for vacuole biogenesis (Larson et al.2013; Latomanski et al.2016; Justis et al.2017). Similarly to S. enterica, C. burnetii metabolism is also induced upon acidification of the CCV; however, the mechanisms behind the activation of C. burnetii metabolism by low pH remain to be elucidated. It is possible that the interactions with autophagosomes are required to deliver nutrients to the otherwise impermeable CCV (Heinzen et al.1996; Gutierrez et al.2005). In addition, proton gradients may facilitate the transport of metabolites and/or that gene transcription is under the regulation of pH sensors (Omsland et al.2008). Interestingly, vacuolar acidification is also required to trigger effector protein translocation by the C. burnetii Dot/Icm T4SS, as illustrated by chemically or genetically perturbing endosomal maturation (Newton, McDonough and Roy 2013). Of note, C. burnetii is the only vacuolar bacterium that is capable of surviving and replicating within a degradative compartment (Howe et al.2010). How C. burnetii resists to lysosomal degradation remains unclear; however, this does not require bacterial metabolism nor the translocation of effector proteins as chloramphenicol-treated bacteria and dot/icm mutants remain viable within lysosomes (Howe et al.2003; Beare et al.2011). Figure 3. View largeDownload slide ‘Friend or foe’, the diverse interactions between vacuolar pathogens and the autophagy machinery. All vacuolar pathogens discussed in this review interact with the autophagy machinery. Some of these interactions however benefit vacuole biogenesis and/or bacterial replication (blue background), whereas others are detrimental to the infectious process (red background). Francisella blocks ATG5-mediated autophagy by a yet unidentified effector protein. However, upon rupture of Francisella-containing vacuoles (FCVs), cytosolic bacteria can be captured by an ATG5-independent autophagy mechanism to generate a non-degradative replicative niche. Ehrlichia translocates the effector protein Etf-1, which interacts with Beclin1, Rab5 and Vps34 to recruit autophagosomes to the replicative niche. Similarly, Anaplasma translocates the effector protein Ats-1, which also interacts with Beclin-1 to recruit autophagosomes to Anaplasma-containing vacuoles. Coxiella translocates the effector protein CvpB/Cig2 which perturbs the activity of the PI3-kinase PIKfyve, thus favouring the autophagy-mediated homotypic fusion of Coxiella-containing vacuoles (CCVs). Also, autophagy is involved in membrane repair following CCVs damage. Upon maturation of Brucella-containing vacuoles (BCVs) to replicative BCVs (rBCVs) autophagy is recruited by an unknown effector protein for the generation of autophagic BCVs (aBCVs), which facilitate the transmission of the pathogen to bystander cells. Autophagy is also involved in membrane repair of Salmonella-containing-vacuoles (SCVs) following T3SS-mediated damage. Conversely, autophagy is also involved in clearance of ubiquitin-tagged (Ub) cytoplasmic Salmonella upon rupture of the SCV. Similarly, autophagosomes are recruited to Mycobacterium-containing vacuoles upon membrane damage induced by ESX-1. Vacuoles subsequently fuse with lysosomes, leading to bacterial degradation. Finally, Legionella translocates the effector proteins RavZ, Lpg1137 and LpSpl to block autophagy and the recruitment of autophagosomes to the Legionella-containing vacuole (LCV). Au, autophagosomes; Ly, lysosomes. Figure 3. View largeDownload slide ‘Friend or foe’, the diverse interactions between vacuolar pathogens and the autophagy machinery. All vacuolar pathogens discussed in this review interact with the autophagy machinery. Some of these interactions however benefit vacuole biogenesis and/or bacterial replication (blue background), whereas others are detrimental to the infectious process (red background). Francisella blocks ATG5-mediated autophagy by a yet unidentified effector protein. However, upon rupture of Francisella-containing vacuoles (FCVs), cytosolic bacteria can be captured by an ATG5-independent autophagy mechanism to generate a non-degradative replicative niche. Ehrlichia translocates the effector protein Etf-1, which interacts with Beclin1, Rab5 and Vps34 to recruit autophagosomes to the replicative niche. Similarly, Anaplasma translocates the effector protein Ats-1, which also interacts with Beclin-1 to recruit autophagosomes to Anaplasma-containing vacuoles. Coxiella translocates the effector protein CvpB/Cig2 which perturbs the activity of the PI3-kinase PIKfyve, thus favouring the autophagy-mediated homotypic fusion of Coxiella-containing vacuoles (CCVs). Also, autophagy is involved in membrane repair following CCVs damage. Upon maturation of Brucella-containing vacuoles (BCVs) to replicative BCVs (rBCVs) autophagy is recruited by an unknown effector protein for the generation of autophagic BCVs (aBCVs), which facilitate the transmission of the pathogen to bystander cells. Autophagy is also involved in membrane repair of Salmonella-containing-vacuoles (SCVs) following T3SS-mediated damage. Conversely, autophagy is also involved in clearance of ubiquitin-tagged (Ub) cytoplasmic Salmonella upon rupture of the SCV. Similarly, autophagosomes are recruited to Mycobacterium-containing vacuoles upon membrane damage induced by ESX-1. Vacuoles subsequently fuse with lysosomes, leading to bacterial degradation. Finally, Legionella translocates the effector proteins RavZ, Lpg1137 and LpSpl to block autophagy and the recruitment of autophagosomes to the Legionella-containing vacuole (LCV). Au, autophagosomes; Ly, lysosomes. Bacteria of the genus Brucella are characterised by an intracellular lifestyle that includes both maturation of BCVs along the endocytic pathway and fusion with host cell organelles (Pizarro-Cerda et al.1998; Celli et al.2003; Celli, Salcedo and Gorvel 2005; Starr et al.2008) (Fig. 2). Upon bacterial internalisation, BCVs interact with early and late endosomes, as indicated by the presence of typical markers of these vesicular compartments at the BCVs (Pizarro-Cerda et al.1998). Despite Brucella sensitivity to lysosomal degradation, BCVs also partially fuse with lysosomes as the expression of the virB T4SS operon is induced at low pH (Boschiroli et al.2002; Starr et al.2008). Translocation of effector proteins allows Brucella to do something remarkable, which is to switch gears from the endocytic pathway to the secretory pathway. Indeed, by a mechanism that remains to be fully elucidated, BCVs interact with ER exit sites (ERES) and acquire ER membranes (Celli, Salcedo and Gorvel 2005; Myeni et al.2013). Furthermore, it has been recently reported that Golgi-derived vesicles participate to BCVs biogenesis (Miller et al.2017) (Fig. 2). Surprisingly though, further maturation of BCVs also involves the subversion of the autophagy pathway as illustrated by the appearance of multi-layered replicative vacuoles that are positive for the autophagy marker LC3 (Starr et al.2012) (Fig. 2, 3). Inhibition of the autophagy machinery correlates with a reduced cell-to-cell transmission of Brucella, suggesting that subversion of autophagy is required at the latest stage of BCV maturation (Starr et al.2012). Accordingly, conditional expression of the VirB11 ATPase demonstrated a role of the Brucella T4SS in the transition from rBCVs to aBCVs and the following cell-to-cell transmission in bone marrow-derived macrophages (Smith et al.2016). Finally, infection of human trophoblasts with Brucella suis and B. abortus revealed alternative replicative niches for these bacteria. Indeed, these pathogens replicate in large, acidic inclusions that fail to acquire ER markers but remain positive for the lysosomal markers LAMP1 and CD63 (Salcedo et al.2013). Whether this represents an adaptation of Brucella to less permissive cells remain to be defined. In favour of this hypothesis, B. melitensis retains the ability of replicating within ER-positive BCVs in extravillous trophoblasts, suggesting that this species can overcome the restrictions imposed by these cells (Salcedo et al.2013). Interaction and fusion of bacteria-containing vacuoles with host cell organelles, which are typical of L. pneumophila and Chlamydia species, favours the biogenesis of replicative compartments characterised by neutral luminal pH. Legionella pneumophila uses a Dot/Icm T4SS apparatus to deliver an astonishing number of effector proteins into the cytosol of infected cells. These manipulate several host cell processes, mediating multiple interactions with the endosomal and the secretory membrane trafficking pathways, culminating with the association of LCVs with the ER (Horwitz 1983; Swanson and Isberg 1995; Robinson and Roy 2006) (Fig. 2). LCVs escape the endosomal maturation pathway early after bacterial internalisation, likely due to the activation of the Dot/Icm secretion system upon contacting the host plasma membrane during phagocytosis (Hubber and Roy 2010). Thus, LCVs acquire ER markers such as KDEL and calnexin (Lu and Clarke 2005) and are anchored to microtubules. LCVs interactions with the ER also occur at ERES and require the activity of host small GTPases including Rab1, Arf1 and Sar1 (Kagan and Roy 2002; Derré and Isberg 2004; Kagan et al.2004). ER-LCV contacts are mediated by the non-canonical pairing between plasma membrane-derived t-SNAREs (syntaxins, SNAP23) on LVCs (acquired upon phagocytosis) and ER v-SNARE Sec22b (Arasaki and Roy 2010). Legionella escapes autophagosomal degradation by translocating effector proteins with protease activity. Lpg1137 is a serine protease specifically targeting Syntaxin 17, leading to the inhibition of omegasome formation (Arasaki et al.2017); the cysteine protease RavZ irreversibly cleaves lipidated LC3 (Choy et al.2012) and LpSpl is a sphingosine-1 phosphate lyase that reduces sphingosine levels in infected cells, thus blocking autophagy (Rolando et al.2016) (Fig. 3). Moreover, LCVs are decorated with small GTPases of the Rab family (Fig. 2), Rap1 and Ran, many of which are implicated in intracellular replication of L. pneumophila (Urwyler et al.2008; Rothmeier et al.2013; Hoffmann et al.2014; Schmölders et al.2017). Recent reports illustrate the role of the host dynamin-like, large GTPase atlastin3 (Atl3) and reticulon 4 (Rtn4) in membrane fusion between the LCV and the ER, which is essential for vacuole expansion and bacterial replication (Kotewicz et al.2017; Steiner et al.2017). The Sde family of L. pneumophila effector proteins, and in particular SdeC, directly targets and ubiquitinates Rtn4 by a sequential ADP-ribosyltransferase and nucleotidase activity encoded by the bacterial effector, thus independently of the ubiquitination machinery of the host (Kotewicz et al.2017). Similarly to L. pneumophila, bacteria of the genus Chlamydia also use translocated effector proteins to escape the endocytic maturation pathway early after internalisation to generate a replicative niche called inclusion, which is characterised by a neutral pH (Heinzen et al.1996) (Fig. 2). In this case, however, the presence of the motor protein dynein at the inclusion membrane mediates the microtubule-driven relocation of the inclusion next to the microtubule organising centre (MTOC), possibly facilitating interactions with the Golgi apparatus (Grieshaber, Grieshaber and Hackstadt 2003). En route to the MTOC, the inclusion acquires a surprising number of Rab GTPases (see below), which is indicative of the complex network of interactions between the Chlamydia replicative niche and membrane trafficking pathways (Fig. 2). Interestingly, certain Rabs associate with the inclusion in a species-specific manner. Thus, Rab1, 4 and 11 are found on inclusions generated by all chlamydial species, whereas Rab6 and Rab10 associate with Chlamydia trachomatis and Ch. pneumoniae, respectively (Rzomp et al.2003). As mentioned above, the characterisation of the inclusion proteome identified 351 proteins and reported an enrichment in sorting nexins, cytosolic proteins capable of interacting with phospholipids and sensing membranes with high curvature. In eukaryotes, these proteins that take part in the retromer, a multiprotein complex recycling transmembrane receptors to the trans-Golgi network (Aeberhard et al.2015). Interestingly, RNAi-mediated depletion of SNX5 resulted in an increase of the infectious progeny, suggesting that the retromer might restrict bacterial replication (Aeberhard et al.2015). Homotypic fusion of Chlamydia inclusions requires the bacterial effector protein IncA and is promoted by microtubule-mediated membrane traffic (Hackstadt et al.1999; Richards, Knowlton and Grieshaber 2013). A hallmark feature of Chlamydia intracellular replication is the acquisition of host-derived lipids (sphingolipids, glycerosphingolipids and cholesterol), which are obtained by re-routing Golgi-derived vesicles to the Chlamydia inclusion (Scidmore, Fischer and Hackstadt 1996; Carabeo, Mead and Hackstadt 2003; Su et al.2004). Of note, the identification of glycerophospholipids, including phosphatidylcholine and phosphatidylinositol, integrated within bacterial membranes indicates that host-derived lipids are transferred from the membranes of the inclusion to Chlamydia (Hackstadt, Scidmore and Rockey 1995; Hackstadt et al.1996; Scidmore, Fischer and Hackstadt 1996; Wylie, Hatch and McClarty 1997). BUILDING BLOCKS: HOST PATHWAY SUBVERSION BY PATHOGENS One of the most effective ways of stalling endosomal maturation is to interfere with the way eukaryotic cells recognise and dispatch vesicles along the many membrane trafficking highways. The identity of most membrane-bound compartments is essentially encoded in their lipid and Rab GTPases composition, which defines what has been earlier referred to as the ‘membrane code’ (Jean and Kiger 2012). Together, PIs and Rab GTPases control the recruitment of host effector proteins coordinating membrane trafficking, fission and fusion events (Jean and Kiger 2012). PIs are key players in the regulation of membrane trafficking (De Matteis and Godi 2004). Their remarkable flexibility in signal transduction relies on the phosphoinositol ring, which can be reversibly phosphorylated at positions 3, 4 and 5, thus generating seven different PI species (De Matteis and Godi 2004). Specific kinases and phosphatases rapidly modify PIs, thereby controlling their distribution in space and time. Lipid metabolism allows the precise and local modulation of essential cellular processes including endocytosis and phagocytosis, membrane tethering, fusion and fission and autophagosome formation (De Matteis and Godi 2004). It is therefore not surprising that PIs and PIs metabolism are targeted by a growing number of cytosolic and vacuolar bacterial pathogens (Pizarro-Cerdá and Cossart 2004; Pizarro-Cerdá, Kühbacher and Cossart 2015). Bacterial effector proteins translocated by vacuolar pathogens can directly bind PIs for membrane targeting and anchoring. This is the case for the L. pneumophila effector proteins SidC, SidM, LidA, LpnE and RidL, which use PI(3)P and/or PI(4)P as anchors to localise at the surface of the LCV and favour interactions with the secretory pathway and the ER (Hilbi, Weber and Finsel 2011) (Fig. 2). Upon LCV localisation, RidL binds the Vps29 retromer subunit and displaces the Rab7 GTPase-activating protein (GAP) TBC1D5, thus blocking endosomes-to-Golgi traffic (Finsel et al.2013). SidM activates the small GTP-ase Rab1 (Machner and Isberg 2006) (see below); LidA interacts with ampylated Rab1, favouring interactions between LCVs and the ER (Machner and Isberg 2006); LpnE recruits the host PI 5-phosphatase OCRL to the Legionella replicative niche (Weber, Ragaz and Hilbi 2009). OCRL is also actively recruited at chlamydial inclusions by means of a yet unidentified Chlamydia effector protein (Moorhead et al.2010). In both cases, OCRL recruitment has been associated with the presence of PI(4)P at LCVs and inclusions (Weber, Ragaz and Hilbi 2009; Moorhead et al.2010). Interestingly, however, inhibition of OCRL has opposite effects on the intracellular fate of these two pathogens: Legionella growth is enhanced by OCRL depletion, suggesting that the phosphatase restricts bacterial growth, whereas optimal biogenesis of the chlamydial inclusion requires OCRL (Weber, Ragaz and Hilbi 2009; Moorhead et al.2010). Bacteria effector proteins can also manipulate PI metabolism either directly, by means of eukaryotic-like kinases and phosphatases secreted by the pathogen, or indirectly, by modulating the recruitment of host PI-metabolising enzymes (Pizarro-Cerdá and Cossart 2004). Collectively, manipulating the lipid profile of their host-derived replicative niche represents an effective method to modulate the interactions between bacteria-containing vacuoles and the endosomal maturation pathway and to ensure optimal intracellular replication (Fig. 2). Consequently, vacuolar pathogens that escape the endocytic maturation pathway early after internalisation tend to exclude PIs that facilitate the intersections with this pathway (Fig. 2). For example, M. tuberculosis depletes its replicative niche of PI(3)P, in order to block phagosomal maturation and avoid fusion with degradative compartments. This is achieved via the PI analogue mannose-capped lipoarabinomannan (ManLAM), which blocks the activity of the type III PI 3-kinase Vps34 (Fratti et al.2003), the secretion of the PI(3)P phosphatase SapM (Vergne et al.2005) and of the broader range phosphatase MptpB (Beresford et al.2007). Reducing the levels of PI(3)P impairs the recruitment of PI(3)P-binding fusion-promoting molecules such as EEA1 or Hrs, which are in turn essential for phagosome maturation (Fratti et al.2003; Vieira et al.2004). Similarly, L. pneumophila reduces the levels of PI(3)P at the LCV to escape the endocytic maturation pathway. This is achieved by means of the secreted effector protein SidP, which hydrolyses PI(3)P and PI(3,5)P2 (Toulabi et al.2013). In addition, the translocated effector protein SidF acts as a PI phosphatase whose activity leads to the accumulation of PI(4)P at the LCV (Hsu et al.2012) (Fig. 2). On the contrary, vacuolar pathogens that follow the endocytic pathway seeking the acidification of their replicative niche tend to facilitate the accumulation of PI(3)P at vacuolar membranes. To this aim, S. Typhimurium uses the SPI-1 substrate SopB, a pleiotropic PI polyphosphatase (Hernandez et al.2004; Mallo et al.2008). Besides promoting Salmonella uptake by acting at the plasma membrane during the early stages of infection, SopB also maintains high levels of PI(3)P and induces Rab5 recruitment to the surface of nascent SCVs, ultimately leading to the formation of large SCVs (Hernandez et al.2004; Mallo et al.2008) (Fig. 2). PI(3)P-binding effector sorting nexin-1 (SNX1) localises to SCVs in a SopB-dependent manner, promoting their remodelling and fostering communication with late endosomal compartments that characterise the later stages of SCV development (Bujny et al.2008). Along the same lines, the C. burnetii effector CvpB binds PI(3)P, thus perturbing the activity of PIKfyve, a PI 3-kinase which plays a key role in early endosomes maturation to late endosomes by phosphorylating PI(3)P to PI(3,5)P2 (Martinez et al.2016). PIKfyve inhibition enriches PI(3)P at CCVs (Martinez et al.2016) and sustains the autophagy pathway (Martin et al.2013), which is required for the homotypic fusion of CCVs (McDonough et al.2012; Newton et al.2014) (Figs 2 and 3). Thus, vacuolar pathogens interfere with lipid metabolism to camouflage their replicative niche and sidetrack it from the lysosomal degradation pathway. It is important to notice that many of these PI-binding bacterial effector proteins have evolved specific PI-binding domains that share no homology with those found in eukaryotes. Together with PIs, Rab GTPases play an essential role in defining the identity of intracellular compartments (Zerial and McBride 2001; Jean and Kiger 2012). Differently from PIs, Rab GTPases cycle between cytosol and cellular membranes, where they stably insert into the outer leaflet upon prenylation (Gomes et al.2003). Similar to PI-mediated signalling however, Rabs act as molecular switches transducing signals at topologically defined locations (Zerial and McBride 2001). Signal transduction depends on the structural conformation of Rabs, which in turn is tightly regulated by their active GTP- or inactive GDP-bound form (Zerial and McBride 2001; Hutagalung and Novick 2011). Specific guanine nucleotide exchange factors (GEFs) and GTP-activating proteins (GAPs) tightly regulate Rabs activity by loading GTP or facilitating its hydrolysis (Zerial and McBride 2001), which is reminiscent of the activity of PI kinases and phosphatases in the regulation of PI signal transduction. If on one hand the topological distribution of PIs is linked to their phosphorylation/dephosphorylation cycle, the human genome encodes over 60 Rab proteins, regulating specific steps of vesicles and organelles dynamics (Hutagalung and Novick 2011). Finally, it is important to note that several steps of intracellular membrane transport are controlled by the coordinated activity of PIs and Rabs (Jean and Kiger 2012). In the context of infections, investigating the ‘Rab signature’ of bacteria-containing vacuoles provides essential information to trace back the biogenesis of bacterial replicative niches and identify the host factors required for their generation. To this aim, proteomic characterisation of intracellular replicative niches has been achieved by isolating Legionella vacuoles and Chlamydia inclusions by immunomagnetic separation and Salmonella vacuoles by cell fractionation (Urwyler et al.2008; Hoffmann et al.2014; Aeberhard et al.2015; Santos et al.2015; Schmölders et al.2017). Besides their biological interest, this information may be used to design host-targeted antimicrobials to interfere with intracellular replication of bacteria. Over 500 proteins have been identified from isolated LCVs, including known host and bacterial components but also Rab GTPases such as Rab1, Rab8 and Rab14 (Urwyler et al.2008) (Fig. 2). Legionella pneumophila directly manipulates Rab GTPases to escape lysosomal degradation early after internalisation. The effector protein SidM uses the pool of PI(4)P generated by the phosphatase activity of SidF as an anchor to localise at LCVs (Brombacher et al.2009). There it sequesters and activates Rab1, the main regulator of the early secretory pathway. Rab1 activation is triggered by a specific GEF activity of SidM, via a GEF domain that shares no homology with other eukaryotic GEFs (Machner and Isberg 2006). Other effector proteins actively manipulate Rab1: AnkX (phosphorylcholination), LepB (GAP), SidD (deAMPylation), Lem3 (dephosphorylcholination) and LidA (Rab1 recruitment on LCV) (Qiu and Luo 2017). Rab1 activation at LCVs is required for fusion events between LCVs and ER membranes, which are mediated by non-canonical interactions between plasma membrane tSNARE syntaxin 3 present on LCVs and the ER vSNARE Sec22b (Kagan et al.2004; Arasaki and Roy 2010). Thus, L. pneumophila triggers fusion events between two intracellular compartments that are topologically distinct outside infection. The activity of SidM is enhanced by another L. pneumophila effector, LidA, which binds a number of Rab GTPases including Rab1, Rab6a and Rab8a but does not present any GEF or GAP activity (Machner and Isberg 2006; Schoebel et al.2009; Mihai Gazdag et al.2013). Interaction of LidA with Rab10 and Rab27a suggests that the effector protein could play a role in the manipulation of the phospholipid content of the LCV and vesicular transport along the secretory pathway (Yu et al.2015). Finally, the effector protein LepB acts as a GAP for Rab1, thus promoting its removal from LCVs (Ingmundson et al.2007). Of note, these effector proteins might have multiple activities as LepB also exhibits GAP activity for Rab3a, Rab8a, Rab13 and Rab35 in vitro. However, whether these functions have in vivo relevance remains to be elucidated. Chlamydia inclusions are characterised by a remarkable number of Rab GTPases (including Rab1, 4, 6, 11, 14, 34, 39), regulating multiple intracellular membrane trafficking steps, which is indicative of the complex biogenesis of this compartment (Damiani, Gambarte Tudela and Capmany 2014) (Fig. 2). Of note, the Golgi-associated Rab14 is required for the delivery of Golgi-derived sphingolipids to the inclusion (Capmany and Damiani 2010). Despite the abundance of Rab proteins identified at chlamydial inclusions, it is important to note that Rab5 and 7, typical markers of the degradative pathway identified in the majority of other bacterial replicative niches, are excluded from these compartments (Rzomp et al.2003) (Fig. 2). Unfortunately, the bacterial factors involved in the regulation of these processes remain largely unidentified, which is mainly due to the difficulties in the genetic manipulation of Chlamydia. Proteomics analysis of SCVs validated the previously reported presence of the late endosomal marker Rab7 and the early endosomal marker Rab5 and revealed the presence of Rab2a, a key regulator of ER-to-Golgi membrane traffic, thus corroborating the observation of SCV/ER MCS at the ultrastructural level (Santos et al.2015). To date, the effector proteins responsible for driving SCV/ER interactions remain to be identified. Of note, an earlier study comparing model phagosomes with SCVs revealed an extended network of Rab GTPases involved in the biogenesis of the Salmonella replicative compartment (Smith et al.2007). Some Rabs were found to be excluded from SCVs, including Rab8, 13, 23, 32 and 35, whereas other Rabs including Rab5, 7, 11 and 14 are enriched at SCVs (Smith et al.2007) (Fig. 2). During vacuole biogenesis, Rab5 and its associated effectors are retained at the surface of the bacterial replicative niche, thus delaying the maturation of SCVs along the endocytic pathway. This is achieved via the combined activity of the SPI-1 effector proteins SopB and SopE (Mukherjee et al.2001; Hernandez et al.2004; Mallo et al.2008). The phosphatase activity of SopB depletes PI(3,5)P2, PI(3,4,5)P3 and PI(4,5)P2 from SCV membranes leading to the recruitment of the PI 3-kinase Vps34 and the generation of PI(3)P, the membrane anchor for Rab5 (Mallo et al.2008) (Fig. 2). Upon Rab5 recruitment at SCVs, the Rab5-specific GEF activity of SopE promotes fusion with early endosomes (Mukherjee et al.2001). In addition, upon activation of the SPI-2 secretion system by vacuolar acidification, the effector protein SopD2 is translocated in the cytosol of the host cell where it binds to and inhibits the activation of Rab7 (D’Costa et al.2015). This perturbs the recruitment of the Rab7 effector proteins RILP (Rab-interacting lysosomal protein) and FYCO1 (FYVE and coiled-coil domain-containing protein 1), thereby disrupting the regulation of microtubules-associated motor proteins, which are required for lysosomal biogenesis (D’Costa et al.2015). The above-mentioned exclusion of Rab32 from SCVs harbouring S. enterica serovar Typhimurium is of particular interest as it has been demonstrated that this Rab GTPase is responsible for host restriction of the typhoid fever agent S. enterica serovar Typhi (Spano and Galan 2012). Indeed, the SPI-1 effector protein GtgE, which is absent in S. Typhi, depletes Rab32 from SCVs by triggering the proteolytic cleavage of this small GTPase (Spano and Galan 2012) (Fig. 2). Rab32 controls traffic to lysosome-related organelles and it is likely involved in the delivery of antimicrobial factors to SCVs. Thus, proteolytic cleavage of Rab32 contributes to SCVs detoxification. Ectopic expression of gtgE in S. Typhi enables bacteria belonging to this serovar to deplete Rab32 from SCVs, thus extending their host range (Spano and Galan 2012). Accordingly, siRNA depletion of Rab32 from cells infected by S. Typhi allows bacteria to replicate in normally restrictive cells (Spano and Galan 2012). Finally, the SPI-2 effector protein SifA, which is also involved in the formation of SIFs, contributes to the detoxification of SCVs by forming a stable complex with the host proteins SKIP and Rab9, thereby perturbing the function of the motor protein kinesin, mannose-6-phosphate receptor trafficking and lysosomal function (McGourty et al.2012). Infections by M. tuberculosis provide another example of how bacteria can indirectly manipulate the Rab signature of their replicative niche by manipulating PI metabolism. As illustrated above, M. tuberculosis translocated effector proteins deplete bacteria-containing compartments of PI(3)P, which serves as an anchor for membrane targeting of the fusion-promoting proteins EEA1 and Hrs (Fratti et al.2003; Vieira et al.2004). These specifically bind PI(3)P via their FYVE domains and are required for the transition from early to late endosomes. Thus, MCVs are positive for the early and recycling endosomal markers such as Rab2, 5, 11, 14 and 22, indicative of the early arrest of vacuole maturation (Fig. 2). It has been reported that Rab14 plays a major role in arresting MCVs maturation as RNAi-mediated depletion of the GTPase or the expression of its dominant negative mutant result in the progression of MCVs along the degradative pathway. It is interesting to note that Rab14 seems to play a role in the biogenesis of bacterial replicative niches (Fig. 2): it mediates the delivery of host lipids to the Chlamydia inclusion (Capmany and Damiani 2010), localises at LCVs and restricts L. pneumophila replication (Hoffmann et al.2014), mediates the acidification of SCVs via the effector Nischarin (Kuijl et al.2013). In addition, Rab14 expression is potentially regulated by the C. burnetii effector protein CBU_1314 (Weber et al.2016a); however, a role for Rab14 in CCVs biogenesis remains to be investigated. How Brucella coordinates the recruitment of Rab GTPases at the vacuole remains largely unknown; however, it has been reported that the effector protein RicA interacts with Rab2, which controls ER-to-Golgi trafficking, thereby mediating its recruitment to BCVs (de Barsy et al.2011) (Fig. 2). Despite the higher affinity for GDP-bound Rab2, it seems unlikely that RicA exhibits a GEF activity for this small GTPase (de Barsy et al.2011). HOME DELIVERY: VACUOLAR LIFESTYLE AND ACCESS TO NUTRIENTS Intracellular pathogens rely on host cells for nutrient acquisition, the main sources being amino acids found in proteins, fatty acids found on lipid droplets and carbohydrates found on glycogen (Steele, Brunton and Kawula 2015). Nutritional requirements have been defined, at least in part, for a number of intracellular bacterial pathogens assessing growth defects associated with nutritional mutants (reviewed in Ray et al.2009). Alternatively, the development of an axenic culture medium for host cell-free growth of the obligate intracellular pathogen C. burnetii provides a beautiful example of how multiple approaches can be used to define the nutritional requirements of obligate intracellular pathogens (Omsland et al.2009). These include knowledge of the intracellular replicative niche, bacterial physiology and genome analysis. Systematic testing of buffer compositions coupled to robust assays allows to define bacteria metabolic activity, growth and infectivity (Omsland et al.2009; Omsland, Hackstadt and Heinzen 2013). If on one hand cytosolic bacteria have direct access to these resources, vacuolar pathogens have to develop strategies to import nutrients through vacuolar membranes. To this aim, targeting of host catabolic pathways is triggered by a number of pathogens. Lysosomes contain degradative enzymes that provide a source of amino acids necessary for the growth of intracellular pathogens such as Chlamydia (Ouellette et al.2011). Alternatively, Chlamydia is also capable of intercepting nutrient-rich Golgi-derived vesicles and multivesicular bodies from the recycling pathway (Bastidas et al.2013; Samanta et al.2017). Furthermore, Ch. trachomatis grows in a glycogen-rich vacuole (Gehre et al.2016). Glycogen is a multibranched polysaccharide used for energy storage in cells. Chlamydia is capable of acquiring this nutrient by two means: transport in bulk from the host cytosol and recruitment of the host UDP-glucose transporter SLC35D2 to the membrane of its inclusion followed by branching of monomeric UDP-glucose into glycogen by the bacterial T3-secreted glycogen synthase GlgA (Gehre et al.2016). The targeting of the autophagy machinery by several vacuolar pathogens, including Coxiella, Brucella, Anaplasma, Ehrlichia and Francisella, may also represent an important source of nutrients (Fig. 3). Coxiella vacuole size increases when autophagy is triggered by starvation in infected cells (Gutierrez et al.2005; Romano et al.2007). As illustrated above, recruitment of the autophagy machinery to the vacuole membrane is dependent on the effector protein CvpB/Cig2 (Newton et al.2014; Kohler et al.2016; Martinez et al.2016) (Fig. 3); however, while vacuole morphology is affected by mutations in cvpB/cig2, C. burnetii is still capable of replicating in these vacuoles, strongly suggesting that additional cellular mechanisms are hijacked by the bacterium to acquire nutrients. While C. burnetii vacuole remains acidic during infection, Anaplasma develops in a vacuole with early autophagosomal properties (Niu et al.2012). In this case, inhibition of autophagy suppresses the intracellular proliferation of Anaplasma, highlighting the importance of this catabolic pathway in the development of the bacterium (Niu et al.2012) (Fig. 3). Autophagy is important for another intracellular pathogen, E. chaffeensis. Even though this bacterium develops in a vacuole with early endosomal features, it requires a functional host autophagy machinery to proliferate. During the course of infection, E. chaffeensis secretes Etf-1, an effector protein that interacts with Rab5, Vps34 and Beclin 1 to induce the Rab5-regulated autophagy pathway (Lin et al.2016) (Fig. 3). Thus, host cell nutrients may be captured by Etf-1-induced autophagosomes and delivered to E. chaffeensis inclusions before their fusion with lysosomes (Lin et al.2016). Francisella tularensis evades xenophagy but increases the flux of ATG5-independent autophagy (Steele et al.2013) (Fig. 3). Of note, this is the only case where autophagy induction has been directly linked to nutrient acquisition by using radiolabelled amino acids (Steele et al.2013). Finally, Chlamydia, Coxiella, Anaplasma and Ehrlichia also manipulate host cholesterol trafficking pathways to access nutrient-rich compartments (Samanta et al.2017). Pathogens that cannot stand the degradative conditions encountered in lysosomes and autolysosomes developed alternative ways to acquire nutrients from the cell. For example, L. pneumophila boosts polyubiquitination of host cell proteins via AnkB and the eukaryotic SCF1 ubiquitin ligase complex (Price, Richards and Abu Kwaik 2014). This leads to increased proteasomal degradation of host cell proteins which generates a surplus of amino acids that can be transported to the LCVs and used by the bacterium (Price, Richards and Abu Kwaik 2014). Interestingly, L. pneumophila and F. tularensis trigger the upregulation of SLC1A5, a host cell amino acid transporter, which is important for intracellular replication of both pathogens (Wieland et al.2005; Barel et al.2012). If on one hand it has been proposed that SLC1A5 may insert into LCVs to facilitate the transport of amino acids from the cytosol to the vacuolar lumen in Legionella-infected cells, its role in Francisella infections remains to be defined. Finally, Legionella also uses glucose to fuel its central metabolism (Eylert et al.2010). Generation of free glucose in infected cells might be facilitated by the Legionella-secreted eukaryotic-like glucoamylase (GamA) which is capable of degrading glycogen and starch (Herrmann et al.2011). PLAYING HIDE AND SEEK: VACUOLE SENSING BY HOST CELLS As illustrated above, the biogenesis of intracellular replicative niches is a strategy adopted by a number of microbes to ‘hide in plain sight’ from cytosolic immune sensing. Interestingly however, this strategy exposes vacuolar pathogens to the host cell innate immune system. Three main mechanisms for the sensing of vacuolar pathogens have been described: (1) direct sensing of pathogen-derived ligands, (2) indirect sensing of pathogen through their effectors and (3) detection of vacuoles by the sensing of altered ‘self’ (Liehl, Zuzarte-Luis and Mota 2015). The direct sensing of pathogen-derived ligands is based on the direct interaction between immune receptors and pathogen ligands. A well-studied example is that of S. Typhimurium T3SS1 and T3SS2, which cause the activation of signals leading to the assembly of the inflammasome (Liehl, Zuzarte-Luis and Mota 2015), mediated by Toll-like and NOD-like innate immune receptors (TLRs and NLRs, respectively) (Broz, Ohlson and Monack 2012). Upon infection of macrophages, S. Typhimurium lipopolysaccharide (LPS) can be detected by TLR4, leading to proinflammatory cytokines production including interleukin-1β (IL-1β) (Broz, Ohlson and Monack 2012). After vacuole formation, NLR family apoptosis inhibitory proteins (NAIPs) detect effector proteins translocated by the T3SS1 into the host cytosol and trigger the activation of NRLs receptors such as NLRC4 (NOD-LRR-and CARD-containing 4) (Liehl, Zuzarte-Luis and Mota 2015). In turn, these signals will trigger the assembly of the inflammasome that activates the caspase-1 signalling cascade, resulting in the release of cytokines and the pathogen clearance by pyroptosis (Moltke et al.2013). Mice express multiple NAIPs, with NAIP1 and 2 recognising T3SS components and NAIP5 and 6 responding to flagellar components (Miao et al.2006). Humans, however, encode a single functional NAIP with broad specificity (Kortmann, Brubaker and Monack 2015; Reyes Ruiz et al.2017). NLRC4 can also work with NAIPs and together detect the T3SS (Zhao et al.2011). Elimination of S. Typhimurium by pyroptosis has in vivo relevance as orogastrically infected mice deficient for caspase-1 and NLRC4 have a larger load of bacteria in liver, spleen and mesenteric lymph nodes compared to the wild-type mice (Broz, Ohlson and Monack 2012). Pathogen recognition can also be facilitated by transporters embedded in the host-cell-derived membranes of bacteria replicative niches that mediate the delivery of pathogen-derived ligands across the vacuole. Two mechanisms have been described in Salmonella species: NOD2 receptor is activated upon binding to muramyl dipeptide, a peptidoglycan motif translocated across the vacuolar membrane by two host endolysomal transporters, SLC15A3 et SLC15A4 (Nakamura et al.2014). NOD2 activation induces the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signalling pathway, resulting in proinflammatory cytokines production and autophagy (Nakamura et al.2014). Alternatively, it has been suggested that the host IFN-induced p65 guanylate-binding proteins (GBPs), belonging to the dynamin family, can destabilise vacuolar membranes, thus exposing the pathogen to host immune recognition (Meunier et al.2014). Indeed, GBPs expression is triggered upon extracellular detection of Salmonella by TLR4 and it has been shown that more bacteria are released to the cytosol of wild-type infected cells as compared to GBP-deficient cells, suggesting indeed that GBPs contribute to destabilisation of the vacuolar membrane (Meunier et al.2014). GBPs are also involved in cytosolic detection of vacuolar bacteria leading to inflammasome activation by the caspase-11-dependent pathway. Thus, GBPs cause damages to SCV therefore releasing the bacteria to the cytosol where these will be recognised by autophagic receptors and destined to elimination. Moreover, besides extracellular recognition mediated by TLR4, bacterial LPS can be recognised in the cytosol by caspase-11 (Kayagaki et al.2013; Shi et al.2014) leading to inflammasome activation. Recent studies have demonstrated that bacterial outer membrane vesicles (OMVs) could deliver LPS to the host cell cytosol, thus triggering the activation of caspase-11 (Vanaja et al.2016; Finethy et al.2017). The detection of M. tuberculosis vacuoles follows a different signalling pathway: the bacterium early secreted antigen 6 SS1 (ESX-1) T7SS induces damages at the membrane of MCVs, which triggers the secretion of interferon I (IFN-I) (Stanley et al.2007; Manzanillo et al.2012). In addition, M. tuberculosis DNA seems to play an important role in pathogen sensing. In fact, cytosolic DNA can be sensed by DNA receptors IFNγ-inducible 16 and the STING-TBK-IRF3 signalling pathway. Bacterial DNA gains cytosolic access either by ESX-1-mediated secretion, spontaneous release following bacterial lysis or membrane permeabilisation and secretion via OMVs (Ellis and Kuehn 2010; Manzanillo et al.2012). Finally, bacterial DNA might be exposed following the activity of host bacteriolytic enzymes that enter the MCV (Liehl, Zuzarte-Luis and Mota 2015). Host cells can also respond to vacuolar pathogen translocated effector proteins, either by direct recognition or by activating signalling pathways downstream of host immune receptors (Liehl, Zuzarte-Luis and Mota 2015). Indeed, besides their known role as virulent factors that promote pathogen survival, effector proteins can also act as immune elicitors. Once again, S. enterica serovar Typhimurium provides well-described examples for this process. In vitro, the effector proteins SopB, SopE2 and SopE elicit the innate immune response by activating Rho GTPase family members Rac1 and Cdc42, which in turn leads to the activation of the mitogen-activated- kinase and nuclear factor- κB (NF-κB) signalling pathways (Bruno et al.2009). In addition, it has been reported that Rho GTPase activation by S. Typhimurium effector proteins can be sensed by NOD1 and heat shock protein 90 (Hsp90), leading to the activation of the innate immune signalling via RIPK2 (enzyme involved in signalling pathway of the immune system, potent activator of NF-κB and inducer of apoptosis) (Keestra et al.2013). Reduced SopE-mediated inflammation detected in NOD1-deficient mice exposed to Salmonella as compared to wild-type mice supports the in vivo relevance of this signalling pathway (Keestra et al.2013). Additionally, the role of Rho-GTPase-mediated signalling in pathogen sensing extends to cytosolic and extracellular bacterial pathogens, which use secreted toxins to modulate Rho activity. Pathogen-mediated Rho inactivation is detected by the Pyrin inflammasome leading to the activation of an antibacterial inflammatory response (Xu et al.2014). The third mechanism of pathogen recognition involves the sensing of altered self. This involves the detection of membrane perturbations caused by infection. Vacuolar pathogens are capable of radically modifying the structure and composition of host intracellular compartments. This may have severe consequences that can thus lead to the activation of innate immune signalling pathways. As mentioned earlier, bacteria-containing vacuoles can lose their integrity, which is usually a consequence of pathogen-mediated membrane rupture (Mansilla Pareja et al.2017). Membrane integrity is sensed by the galectin family of host proteins (Thurston et al.2012). These are cytosolic proteins that detect physical damage by binding glycans which are usually exposed at the cell surface or at the luminal side of endomembranes (Vasta et al.2012). Upon membrane damage, glycans are exposed to the cytosol of the cell where they can be detected by galectins. For example, SCV membrane rupture during S. Typhimurium infections exposes glycans which are detected by Galectin 8. This will lead to the recruitment of two autophagy adaptor proteins NDP52 and LC3C and autophagy-mediated bacterial clearance (Thurston et al.2012). However, the autophagy machinery has also been reported to repair SCVs damaged by the presence of the T3SS1 (Kreibich et al.2015). A similar mechanism has been recently reported for C. burnetii infections, where Galectin 3 and 8 are recruited at sites of CCVs damage (Mansilla Pareja et al.2017). In this case again, it seems that autophagy contributes to the re-sealing of vacuoles, thus ensuring bacterial replication. Alternatively, infected cells can sense changes in the molecular features that normally characterise a given membrane-bound compartment, without necessarily involving structural damage. This mechanism is mediated by IFN-inducible GTPases of the immunity-related GTPases (IRG), which include the membrane-bound IRGM proteins and the cytosolic GKS proteins (Haldar et al.2013). As indicated above, GKS trigger membrane destabilisation, which can be used as a countermeasure against parasitophorous vacuole biogenesis. On the contrary, IRGMs are negative regulators of GKS activity and are abundant on ‘self’ compartments such as the ER, Golgi apparatus and endolysosomes (MacMicking 2012), which are thus protected from GKS-dependent destabilisation. Despite being derived from host membranes, Ch. trachomatis inclusions only present limited amounts of IRGMs at the surface, making these compartments the target of GKS activity, ultimately releasing bacteria in the cytosol, where they can be cleared by autophagy (Al-Zeer et al.2009; Haldar et al.2013). SHOULD I STAY OR SHOULD I GO: LIFE AS AN INBETWEENER As mentioned earlier, the fine line between cytosolic and vacuolar lifestyles for intracellular bacterial pathogens is becoming blurred. It is now well established that S. enterica serovar Typhimurium can also escape SCVs and is capable of sustained replication within the cytosol of infected cells, albeit only epithelial cells are permissive for cytosolic hyper-replication (Knodler et al.2010) (Fig. 1). Vacuolar escape occurs at early and late steps of the SCV maturation. Early escape seems to be uniquely mediated by the membrane-destabilising properties of the SPI-1 T3SS as ‘effectorless’ mutants retain the ability to escape (Birmingham et al.2006; Knodler, Nair and Steele-Mortimer 2014). Accordingly, SPI-1 genes are highly expressed in cytosolic bacteria (Knodler et al.2010) and the SPI-1 effector proteins SopB and SipA have been shown to be important for cytosolic hyper-replication in an elegant study that uses controlled activation of the SPI-1 injectisome (Klein et al.2017). Later escape may be in part triggered by defects in SCVs membrane stabilisation, which is supported by SPI-2 T3SS effector proteins including SifA (reviewed in Knodler 2015). It has also been reported that early fusion events between SCVs and micropinosomes contribute to SCVs membrane stability, thus promoting intravacuolar replication (Fredlund et al.2017). A mutational analysis identified four Salmonella genes involved in the modulation of cytosolic replication (ydgT, recA, corA and asmA)(Wrande et al.2016). Cytosolic hyper-replication of Salmonella is particularly surprising if we consider the role of autophagy in the innate immune response to cytosolic microbes. It has been reported that indeed, cytosolic replication of Salmonella is partially restricted by autophagy, as shown by depleting autophagy-associated proteins in infected cells (Birmingham et al.2006). However, Salmonella actively inhibit autophagy by restoring the levels of cytosolic amino acids, which in turn favours the activation of the autophagy inhibitor kinase mTOR (Tattoli et al.2012) (Fig. 3). Emerging evidence supports M. tuberculosis escape from MCVs (Stamm et al.2003; van der Wel et al.2007; Simeone et al.2012; 2015). In this case, vacuolar escape occurs at late stages of infection and relies on the ESX-1 type VII secretion system (T7SS) and the outer membrane phthiocerol dimycocerosates, which trigger contact-dependent cell membrane lysis (Augenstreich et al.2017; Conrad et al.2017) (Fig. 1). The T7-secreted protein ESAT-6 (also known as EsxA) has been predicted to induce the formation of membrane pores (Smith et al.2008). However, even though this effector has membrane-destabilising properties (Augenstreich et al.2017), its membranolytic activity in vitro seems to be due to preparation artifacts (Conrad et al.2017). Whether Mycobacterium replicates in the cytosol of the host cell is still unclear; however, the observation that MCV rupture by M. tuberculosis and M. marinum is followed by necrotic cell death suggests that Mycobacterium uses this strategy for dissemination (Stamm et al.2003; van der Wel et al.2007). A recent study reported how the block of MCV acidification and the capacity of Mycobacterium to rupture its vacuole are directly correlated and, more importantly, provided the first in vivo evidence for vacuolar escape in spleen and lungs from mice infected with M. tuberculosis (Simeone et al.2015). Of note, dual intracellular lifestyle goes both ways as it has been reported that, in infected macrophages, the cytosolic bacterium Listeria monocytogenes can also replicate (albeit less efficiently) into spacious Listeria-containing phagosomes, which are positive for the lysosomal marker LAMP1 but are non-acidic and non-degradative (Birmingham et al.2008). Generation of these compartments correlates with the level of expression of the Listeria pore-forming toxin listeriolysin O and involves host autophagy (Birmingham et al.2008). More recently, it has been shown that after several days of infection of epithelial cells, Listeria stops producing the actin-nucleating protein ActA and becomes trapped into Listeria-containing vacuoles (LisCVs) (Kortebi et al.2017) (Fig. 1). Here, a subpopulation of bacteria enters a viable but non-culturable state, insensitive to gentamicin treatment. Over time, these forms were shown to cycle between vacuolar and cytosolic stage, suggesting that the LisCV stage represents a form of persistence for Listeria infections (Kortebi et al.2017). Finally, it has been reported that following early phagosomal escape and cytosolic replication of F. tularensis, bacteria are captured within Francisella-containing vacuoles by an autophagy-mediated process (Checroun et al.2006) (Figs 1 and 3). Morphological characterisation of these compartments showed double-layered membranes typical of autophagosomes and the presence of the autolysosomal markers LC3 and LAMP1 (Checroun et al.2006). Despite interactions with lysosomal compartments, F. tularensis seems capable of resisting degradation, as indicated by the observation of intact bacteria by electron microscopy (Checroun et al.2006). CONCLUDING REMARKS In the course of evolution, interactions between prokaryotes and eukaryotes led to the emergence of bacterial measures and host cell countermeasures which resolved either in symbiosis or parasitism. Intracellular bacterial pathogens developed sophisticated camouflaging mechanisms allowing microbes to invade eukaryotic cells and establish an intracellular replicative niche. These processes are mediated by a beautifully diversified array of bacterial proteins that have evolved to mimic host cell proteins and subvert a variety of cellular functions. In some cases, the evolution of new functional eukaryotic-like domains in bacterial proteins emerged from exchanges in genetic material between prokaryotes and eukaryotes, which is indicative of the intimate connexions existing between these two domains. Phylogenetic trees analysis strongly suggests that horizontal gene transfer from eukaryotes to bacteria occurred during the course of evolution, thus enabling bacteria to manipulate very specific host cell mechanisms. In turn, this has been exploited in the field of cellular microbiology to explore eukaryotic pathways using microbes and/or derived proteins as probes. Among intracellular microbes, vacuolar bacteria have evolved to ‘terraform’ their host by driving the biogenesis of new organelles, which are mainly constituted of host cell membranes, but exist only in the context of a bacterial infection. Upon internalisation, bacteria establish a dialogue with the infected cell, by sending signals in the form of effector proteins, to which the cell responds by rewiring of specific signalling pathways. As illustrated in this review, the biogenesis of a host-cell-derived replicative niche provides sanctuary to bacterial pathogens, away from extracellular and cytoplasmic immune surveillance. It is interesting to see however that, despite this camouflaging technique deployed by bacterial pathogens, host cells have developed countermeasures to detect the formation of aberrant compartments, which activate antimicrobial responses. Bacterial replicative niches are extremely diversified in their morphology, composition and dynamics. More importantly, the biogenesis of different replicative niches results in very different cellular fates, ranging from cell lysis to vacuolar expulsion or persistence. This reflects different degrees of adaptation of a microbe to its host and may suggest that in some cases, pathogens are still evolving towards a symbiotic lifestyle. Despite the great diversity among bacterial effector proteins, it is interesting to note that most vacuolar pathogens target common signalling hubs within host cells. Thus, vacuole biogenesis is achieved by the manipulation of host signalling components that are key regulators of membrane traffic such as Rab GTPases, lipids and phospholipids. The study of bacterial vacuole biogenesis and the identification of the essential building blocks of each replicative niche can be exploited to identify key host factors that could be targeted for the development of new, tailored antimicrobials. Indeed, the emergence of multidrug-resistant pathogens has fostered research towards the development of new antimicrobial and host-targeted therapies may represent a valuable option. 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This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Reviews Oxford University Press

Tiny architects: biogenesis of intracellular replicative niches by bacterial pathogens

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10.1093/femsre/fuy013
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Abstract

Abstract Co-evolution of bacterial pathogens with their hosts led to the emergence of a stunning variety of strategies aiming at the evasion of host defences, colonisation of host cells and tissues and, ultimately, the establishment of a successful infection. Pathogenic bacteria are typically classified as extracellular and intracellular; however, intracellular lifestyle comes in many different flavours: some microbes rapidly escape to the cytosol whereas other microbes remain within vacuolar compartments and harness membrane trafficking pathways to generate their host-derived, pathogen-specific replicative niche. Here we review the current knowledge on a variety of vacuolar lifestyles, the effector proteins used by bacteria as tools to take control of the host cell and the main membrane trafficking signalling pathways targeted by vacuolar pathogens as source of membranes and nutrients. Finally, we will also discuss how host cells have developed countermeasures to sense the biogenesis of the aberrant organelles harbouring bacteria. Understanding the dialogue between bacterial and eukaryotic proteins is the key to unravel the molecular mechanisms of infection and in turn, this may lead to the identification of new targets for the development of new antimicrobials. host/pathogen interactions, vacuolar bacterial pathogens, vacuole biogenesis, membrane traffic HUNTER GATHERERS VERSUS SETTLERS AND THE INBETWEENERS: INTRACELLULAR LIFESTYLES OF BACTERIAL PATHOGENS The development and establishment of strategies allowing bacteria to survive and proliferate inside eukaryotic cells traces back in ancient times and has shaped the evolution of eukaryotic cells as we know them. Bacteria use eukaryotic cells as safe houses, providing sanctuary from a harsh environment, to access nutrients as well as means of transportation. Harbouring bacteria can be either beneficial, neutral or detrimental for eukaryotic cells (Steinert, Hentschel and Hacker 2000). The endosymbiotic theory represents the best example of how interactions between prokaryotes and eukaryotes can be beneficial, to the point where bacteria evolve to become respiratory organelles: mitochondria for animal cells (likely derived from alphaproteobacteria ancestors) (Thrash et al.2011) and chloroplasts for plant cells (possibly related to cyanobacteria) (Falcón, Magallón and Castillo 2010). On the opposite side of the scale, infectious diseases stand as a threatening example of how interactions between bacteria and eukaryotic cells can be deleterious for the latter. The first step towards the establishment of an intracellular lifestyle is the internalisation within a eukaryotic cell. Access to phagocytic cells is passively achieved due to the clearance activity of macrophages. Internalisation within non-phagocytic cells on the other hand is mediated either by bacterial surface proteins, which activate receptor-mediated endocytosis signalling pathways, or by bacterial effector proteins, which are injected into the host cell cytosol to drive the reorganisation of the host cell cytoskeleton and bacterial engulfment. These two processes are commonly referred to as zippering and triggering mechanisms, respectively (Cossart 2004). Despite the numerous advantages associated with an intracellular lifestyle, the cellular environment remains inhospitable for bacteria: inside endosomes, nutrients are scarce and material internalised either by endocytosis or phagocytosis is destined to lysosomal degradation; on the other hand, the cytosol is rich in nutrients, but constantly surveyed by the innate immune system. Bacteria have adapted to this environment by adopting very different lifestyles, reminiscent of those observed in early humans: cytosolic bacteria escape the degradative pathway by rupturing internalisation vacuoles thus accessing the cytosol. Here, bacteria including Listeria monocytogenes, Shigella flexneri, Rickettsia species and Mycobacterium marinum and Burkholderia live like hunters-gatherers, harnessing the actin cytoskeleton to escape the autophagy-mediated innate immune surveillance, gather nutrients and explore the intracellular environment to access bystander cells (Gouin, Welch and Cossart 2005) (Fig. 1). Of note, intracellular motility can also be fuelled by flagellar motors (Knodler et al.2010) or by a combination of the two (French et al.2011). On the other hand, vacuolar bacteria settle within internalisation vacuoles and re-shape cellular compartments and organelles to create new environments, which are derived from eukaryotic components but only exist in the context of bacterial infections (Cossart and Roy 2010) (Fig. 1). As always in nature, this dichotomy is not 100% accurate. Thus, given the right environment or conditions, some cytosolic bacteria can also replicate within membrane-bound compartments and conversely, an increasing number of vacuolar pathogens escape their niche to replicate in the cytosol (Fig. 1). This review will mainly focus on vacuolar bacteria and the many strategies developed to generate pathogen-specific replicative niches exploiting the resources of host cells. Figure 1. View largeDownload slide Intracellular lifestyles of bacterial pathogens. Upon internalisation within host cells, bacteria of the genus Chlamydia, Legionella, Ehrlichia, Anaplasma, Brucella and Coxiella remain confined within their internalisation vacuole, which they modify by driving pathogen-specific interactions with membrane-bound organelles and/or transport carriers to develop a replicative niche. Depending on their adaptation to an acidic environment, vacuolar pathogens can escape the endosomal maturation pathway by acquiring markers of organelles such as the endoplasmic reticulum (Legionella and Brucella) or the Golgi complex (Chlamydia and Brucella). Other microbes including Listeria, Rickettsia, Shigella and Burkholderia species as well as Mycobacterium marinum escape the internalisation vacuole and replicate in the cytoplasm where they can use actin- or flagellar-based motility to escape the innate immune surveillance and infect bystander cells. Finally, Mycobacterium, Salmonella, Francisella and Listeria may adopt a dual lifestyle, which includes both vacuolar and cytoplasmic stages. Inc, inclusion; LCV, Legionella-containing vacuole; BCV, Brucella-containing vacuole; CCV, Coxiella-containing vacuole; MCV, Mycobacterium-containing vacuole; SCV, Salmonella-containing vacuole; FCV, Francisella-containing vacuole; LisCV, Listeria-containing vacuole. Figure 1. View largeDownload slide Intracellular lifestyles of bacterial pathogens. Upon internalisation within host cells, bacteria of the genus Chlamydia, Legionella, Ehrlichia, Anaplasma, Brucella and Coxiella remain confined within their internalisation vacuole, which they modify by driving pathogen-specific interactions with membrane-bound organelles and/or transport carriers to develop a replicative niche. Depending on their adaptation to an acidic environment, vacuolar pathogens can escape the endosomal maturation pathway by acquiring markers of organelles such as the endoplasmic reticulum (Legionella and Brucella) or the Golgi complex (Chlamydia and Brucella). Other microbes including Listeria, Rickettsia, Shigella and Burkholderia species as well as Mycobacterium marinum escape the internalisation vacuole and replicate in the cytoplasm where they can use actin- or flagellar-based motility to escape the innate immune surveillance and infect bystander cells. Finally, Mycobacterium, Salmonella, Francisella and Listeria may adopt a dual lifestyle, which includes both vacuolar and cytoplasmic stages. Inc, inclusion; LCV, Legionella-containing vacuole; BCV, Brucella-containing vacuole; CCV, Coxiella-containing vacuole; MCV, Mycobacterium-containing vacuole; SCV, Salmonella-containing vacuole; FCV, Francisella-containing vacuole; LisCV, Listeria-containing vacuole. TOOLS FOR THE JOB: SECRETION SYSTEMS AND EFFECTOR PROTEINS To establish a communication with their recipient host cell and manipulate their machineries, pathogenic Gram-negative intracellular bacteria developed several secretion systems, which are complex macromolecular nanomachines that span both their inner and outer membranes to permit the translocation of effector proteins from the bacterial cytosol to the host cell cytosol. Among the nine existing secretion system, only type 3, 4 and 6 (T3SS, T4SS and T6SS, respectively) are capable of protein translocation (Costa et al.2015). Each intracellular bacterial pathogen needs to shape its own replicative niche. To do so, these bacteria use type 3 (Salmonella, Chlamydia, Simkania) or type 4 (Brucella, Legionella, Coxiella, Anaplasma, Ehrlichia) secretion systems. Genes encoding effector proteins often possess specific promoter sequences that are co-regulated with genes encoding their respective secretion system. Furthermore, effector proteins often carry N-terminal or C-terminal secretion signals (features including positive charges, basicity, hydrophobicity and secondary structures); eukaryotic localisation signals, which target the bacterial protein to specific host cell compartments; eukaryotic-like domains, which are often involved in the manipulation of host cell functions; and other functional domains (Escoll et al.2016). Thus, bacterial effector proteins can either mimic or modify endogenous proteins to alter host cell pathways (Dean 2011; Pearson et al.2015). The expression and secretion of effector proteins can be triggered by external signals. For example, Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system (T3SS) gene expression is activated by acidic pH, decrease in inorganic phosphate, magnesium and calcium concentrations (Löber et al.2006). In macrophages, this leads to an upregulation of SPI-2-dependent effector genes and a downregulation of the Salmonella pathogenicity island 1 (SPI-1) T3SS-dependent effector genes (Srikumar et al.2015). For Coxiella and Brucella, acidification of their respective bacteria-containing phagosomes activates their metabolism which then triggers T4SS activation and effector secretion (Boschiroli et al.2002; Newton, McDonough and Roy 2013). In the case of Legionella pneumophila, a subset of effector proteins are ‘pre-synthesised’ at later stages of the intracellular cycle, prior to host cell lysis, and translocation is triggered upon contact between the Dot/Icm T4SS and the plasma membrane of bystander cells (Charpentier et al.2009). Given their importance in the infectious cycle and virulence of intracellular bacterial pathogens, the identification and characterisation of effector proteins has fostered extensive research. Bioinformatics analysis of bacterial genomes carrying T3SS (Arnold et al.2009; Tay et al.2010; Hobbs et al.2016) and T4SS (Bi et al.2013; Lifshitz et al.2013; Meyer et al.2013; Wang et al.2014) was used to identify ‘eukaryotic-like’ genes (EUGENs) encoding hypothetical proteins with eukaryotic-like domains and motifs: ankyrin repeats, SEL1 (TPR), Set domain, Sec7, serine threonine kinase domains, U-box, F-box, Src homology 2, pentatricopeptide repeat domains, phosphorylation motifs targeted by host kinases and prenylation domains (Dean 2011; Gomez-Valero et al.2011). Results from these studies were corroborated by experimental data arising from comprehensive genetic screens using adenylate cyclase and beta lactamase fusion assays (Chen et al.2010; Carey et al.2011; Zhu et al.2011). The overall results of bioinformatics and experimental analysis were used in machine learning approaches to further characterise potential effector proteins (Lifshitz et al.2014; Martínez-García, Ramos and Rodríguez-Palenzuela 2015; An et al.2017). First identified in Legionella and Chlamydia (Cazalet et al.2010), EUGENs share a high degree of similarity with eukaryotic proteins, suggesting an initial acquisition, via horizontal gene transfer, from eukaryotic organisms (Lurie-Weinberger et al.2010; Gomez-Valero and Buchrieser 2013). Conversely, other effectors may have arisen from convergent evolution. These carry functional domains that are capable of interacting with and/or modifying eukaryotic targets and that possess no sequence homology with known eukaryotic domains or proteins. Finally, some bacterial effectors combine these two types of domains. Legionella effector SidM/DrrA appears as a prime example of such rearrangement. It possesses three domains: an N-terminal adenosine monophosphate transferase (AMPylase) domain, a central guanosine exchange factor (GEF) domain and a C-terminal phosphatidylinositol-4-phosphate binding domain (P4M). While the AMPylase domain shares structural similarities and an important catalytic motif with known eukaryotic glutamine synthase adenylyl transferases (Müller et al.2010), the GEF and P4M domains are structurally distinct from any know eukaryotic domain with similar functions (Schoebel et al.2010; Suh et al.2010). It is remarkable to observe how translocated effector proteins are unique from one species of intracellular bacteria to another and specifically manipulate host cell mechanisms and pathways to define and expand the adequate niche to support bacterial replication. Salmonella enterica serovars cause a range of diseases in humans from gastroenteritis to typhoid fever. These bacteria possess two T3SS: SPI-1, which is used for triggering internalisation of the bacteria, and SPI-2, which is essential for the biogenesis of Salmonella-containing vacuoles (SCVs). SPI-2 is used to inject 28 effectors in the host cell endomembrane system and cytosol (Figueira and Holden 2012). A remarkable feature of SCVs is the presence of Salmonella-induced tubules that radiate from their surface (Liss and Hensel 2015). Eight effector proteins tightly regulate membrane dynamics of SCVs: SifA, PipB2, SseJ, SopD2, SseF, SseG, SteA and SpvB (Knuff and Finlay 2017). While SifA and PipB2 promote the formation of Salmonella-induced filaments (SIFs), maintain SCVs membrane integrity and enable the continuous fusion of host vesicles to SCV membranes, their action is counteracted by the activities of SseJ, SopD2 and SpvB. Furthermore, SifA forms a complex with SKIP and Rab9, thus inhibiting the Rab9-dependent transport of Mannose 6-phosphate receptor (M6PR) to SCVs (McGourty et al.2012). This decreases the delivery of lysosomal enzymes to SCVs and protects intracellular Salmonella from host defences. Other effector proteins that might participate to SCV biogenesis are PipB, an SCV-interacting effector with no yet defined function (Knodler et al.2003), and GtgE, a protease that cleaves Rab29 and Rab32 off SCVs (Spano and Galan 2012). Chlamydia species are obligate intracellular bacterial pathogens that replicate in a specialised membrane compartment called the inclusion. These bacteria use a T3SS to inject between 36 and 107 effector proteins in the host cytosol, depending on the species (Elwell, Mirrashidi and Engel 2016). Chlamydia trachomatis injects CT229, which binds to Rab4 (Rzomp, Moorhead and Scidmore 2006; Ronzone and Paumet 2013), IncA (CT119), InaC (CT813) and IPAM (CT223) which act as inhibitory SNAREs to limit fusion of the inclusion with VAMP3, VAMP7 and VAMP8-positive compartments (Ronzone and Paumet 2013; Elwell, Mirrashidi and Engel 2016), and IncE (CT116) which binds Sorting Nexins 5, 6 and 32 (SNX5, SNX6 and SNX32 respectively) to disrupt host trafficking pathways and promote bacterial growth (Paul et al.2017). Finally, IncD and IncV effector proteins localise at endoplasmic reticulum (ER)-inclusion membrane contact sites (MCS) and interact with ER-resident proteins CERT, VAPA and VAPB to mediate lipid transfer and inclusion biogenesis (Derré, Swiss and Agaisse 2011). Brucella species cause brucellosis in a wide variety of domestic and wild animals, humans being secondary or accidental hosts. The bacteria uses a T4SS to secrete approximately 15 effector proteins during the course of infection, some of which perturb ER secretion (Myeni et al.2013; Ke et al.2015). Effector protein RicA is capable of interacting with the inactive form of the small Rab GTPase Rab2 and modulates the maturation of Brucella-containing vacuoles (BCVs) (de Barsy et al.2011). Another effector protein favouring the biogenesis of BCVs is SepA (Döhmer et al.2014). While the function of the protein has not been established in detail, its secretion during the early stages of infection abolishes the fusion of lysosomes with BCVs, thus favouring the establishment of a suitable replicative niche for the bacterium. Furthermore, it has been recently shown that BspB is capable of interacting with the conserved oligomeric Golgi (COG) tethering complex to redirect Golgi-derived vesicles to the BCV (Miller et al.2017). Coxiella burnetii, the causative agent of Q fever, translocates approximately 133 effector proteins (Moffatt, Newton and Newton 2015; Larson et al.2016; Qiu and Luo 2017). Bacteria develop a unique spacious, acidified vacuole (the Coxiella-containing vacuole, CCV) containing active hydrolases. Five C. burnetii effector proteins have been shown to localise at CCVs and have thus been referred to as Coxiella vacuolar proteins (CvpA to E) and mutations in cvp genes severely affect CCVs biogenesis and C. burnetii replication (Larson et al.2015). Despite the fact that their mode of action remains largely elusive, it has been reported that CvpA binds AP2 adaptor complexes on recycling endosomes, re-routing these vesicles to the CCV (Larson et al.2013) and CvpB inhibits the activity of the PI 3-kinase PIKfyve to maintain vacuolar phosphatidylinositol 3-phosphate (PI(3)P), which is used for homotypic vacuole fusion via the autophagy machinery (Newton et al.2014; Kohler et al.2016; Martinez et al.2016). In addition, Cig57 interacts with components of the clathrin-mediated vesicular trafficking and is required for vacuole biogenesis (Latomanski et al.2016) and CirA recruits and stimulates the GTPase activity of RhoA at the CCV (Weber et al.2016b). A Coxiella evolutionary related pathogen is L. pneumophila, the causative agent of Legionnaire's disease. This bacterium also possesses a T4SS whose structure has recently been characterised (Ghosal et al.2017). At least 330 effectors have been identified for this pathogen and approximately 40 of these have been linked to the manipulation of vesicle identity and trafficking to favour the biogenesis of Legionella-containing vacuoles (LCVs) (Qiu and Luo 2017). At least six effector proteins play a role in the recruitment, stabilisation and recycling of the small GTPase Rab1 on LCVs: SidM/DrrA, AnkX, LepB, SidD, Lem3 and LidA (Qiu and Luo 2017). Additionally, the effector protein RalF functions as an ARF1 GEF on LCVs (Nagai et al.2002). Activated ARF1 may promote recycling of coat proteins from the LCV back to the ER, thus favouring the expansion of the LCV (Nagai et al.2002). Other effector proteins participate in the biogenesis of LCVs include VipD, a Rab5-interacting effector with a phospholipase A1 activity that inactivates PI(3)P on early endosomes, thus changing their lipid composition and inhibiting their fusion with LCVs (Gaspar and Machner 2014). The effector protein SidK blocks the acidification of LCVs by inhibiting the activity of the vacuolar ATPase (v-ATPase) that pumps protons across membranes (Xu et al.2010). Finally, RavZ is a secreted cysteine protease that inhibits host autophagy by selectively deconjugating LC3 from host membranes (Choy et al.2012). Mycobacterium tuberculosis is a facultative intracellular pathogen responsible for millions of casualties each year. Following internalisation, the bacterium secretes two effector proteins that are capable of arresting the maturation of Mycobacterium-containing vacuoles (MCVs). Mycobacterium secretes the acid phosphatase SapM, which has strong affinity for PI(3)P and removes this signalling lipid from MCVs, thus blocking the phagosomal maturation during infection (Saleh and Belisle 2000). Ndk is a nucleoside diphosphate kinase that inactivates the GTPases Rac1, Rab5 and Rab7. Thus, Ndk not only triggers a decrease in lysosomal fusion with bacteria-containing phagosomes but also a decrease in the production of reactive oxygen species that would be deleterious to Mycobacterium replication (Sun et al.2010, 2013). Table 1 provides a complete list of the above-mentioned effector proteins, their eukaryotic targets and function in the regulation of membrane traffic and vacuole biogenesis. Table 1. Bacterial effector proteins participating in vesicle trafficking and vacuole biogenesis, their eukaryotic targets and main function. Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) View Large Table 1. Bacterial effector proteins participating in vesicle trafficking and vacuole biogenesis, their eukaryotic targets and main function. Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) Effector Interactor or target Activity Function Reference Anaplasma Ats-1 Beclin 1 Nucleates autophagosomes Recruits the autophagosomal initiation machinery to Anaplasma inclusion membrane Niu et al. (2012) Brucella BspB Conserved oligomeric Golgi tethering complex (COG) Unknown Redirects Golgi-derived vesicles to the BCV Miller et al. (2017) RicA Rab2 Unknown Modulation of BCV maturation de Barsy et al. (2011) SepA Unknown Unknown Alters LAMP-1 dynamics on the BCV Döhmer et al. (2014) Chlamydia CT229 Rab4 Unknown Binds active Rab4 Rzomp, Moorhead and Scidmore (2006) CT850 Dynein light chain 1 subunit DYNLT1 Unknown Participates in the positioning of the inclusion at the MTOC Mital et al. (2015) InaC (CT813/CTL0184) 14–3-3 β, 14–3-3 ε, ARF1 and ARF4 Unknown Modulates actin assembly and Golgi positioning around the inclusion Kokes et al. (2015); Wesolowski et al. (2017) IncA Unknown SNARE-like protein Inhibits endocytic SNARE machinery, triggers homotypic fusion of the inclusion Delevoye et al. (2008); Paumet et al. (2009); Weber et al. (2016c) IncD (CT115) CERT Unknown Recruits the lipid transfer protein CERT and the ER-resident protein VAPB to ER-inclusion membrane contact sites Derré, Swiss and Agaisse (2011); Agaisse and Derré (2014) IncE SNX5, SNX6 and SNX32 Unknown Disrupts retromer trafficking Mirrashidi et al. (2015); Elwell et al. (2017); Paul et al. (2017) IncV VAPA and VAPB Unknown Acts as a molecular tether to promote the formation of ER-inclusion membrane contact sites Stanhope et al. (2017) Coxiella Cig57 (CBU_1751) FCHO2 Unknown Subversion of clathrin-mediated vesicle trafficking Latomanski et al. (2016) CirA (CBU_0041) RhoA Stimulates GTPase activity of RhoA Recruitment of Rho GTPase to promote CCV biogenesis Weber et al. (2016b) CvpA (CBU_0665) Clathrin adaptor AP2 Unknown Subversion of clathrin-mediated vesicle trafficking Larson et al. (2013) CvpB/Cig2 (CBU_0021) PI(3)P and PS Interferes with PIKfyve activity Triggers recruitment of LC3 to CCVs and homotypic fusion of CCVs Kohler et al. (2016); Martinez et al. (2016) CvpC (CBU_1556) Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpD Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) CvpE Unknown Unknown Localises to and participates in CCVs biogenesis Larson et al. (2015) Ehrlichia Etf-1 (ECH0825) Rab5, PIK3C3/VPS34 and Beclin 1 Activates class III PtdIns3K Increases cellular PI(3)P and induces autophagosome formation Lin et al. (2016) Legionella AnkX (LegA8/Lpg0695) Rab1 and Rab35 Phosphocholinase Modulation of Rab1 and Rab35 activity Mukherjee et al. (2011); Tan, Arnold and Luo (2011) Ceg9 (Lpg0246) Reticulon 4 (Rtn4) Unknown Subversion of vesicle trafficking and ER tubulation Haenssler et al. (2015) Ceg19 (Lpg1121) Unknown Unknown Subversion of vesicle trafficking Heidtman et al. (2009) LecE (Lpg2556) Unknown Activator of phosphatidic acid phosphatase Pah1 Manipulation of host phospholipids metabolism Viner et al. (2012) LegC2 (YlfB/Lpg1884) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegC3 (Lpg1701) Unknown Unknown Subversion of vesicle trafficking de Felipe et al. (2008); Bennett et al. (2013) LegC7 (YifA/Lpg2298) Unknown SNARE-like protein Subversion of ER-derived vesicle trafficking, enhance the efficiency of LCV remodelling Campodonico, Roy and Ninio (2016) LegK2 (Lpg2137) ARPC1B and ARP3 Kinase Interferes with late endosome/lysosome trafficking to the LCV, inhibits actin polymerisation on the LCV membrane Michard et al. (2015) Lem3 (Lpg0696) Rab1 and Rab35 Dephosphocholinase Modulation of Rab1 and Rab35 activity Tan, Arnold and Luo (2011); Goody et al. (2012) LepB (Lpg2490) Rab1, Rab3, Rab8, Rab13 and Rab35 GAP Modulation of Rab GTPases activity Ingmundson et al. (2007); Mihai Gazdag et al. (2013) LidA (Lpg0940) Rab1, Rab6A΄, Rab8, PI(3)P and PI(4)P GTPase stabiliser Binds active Rab1, Rab6A΄ and Rab8 Schoebel et al. (2011); Neunuebel et al. (2012); Chen and Machner (2013) LpdA (Lpg1888) Unknown Phospholipase D Produces diacylglycerol and phosphatidic acid, subverts host phospholipid biosynthesis Viner et al. (2012); Schroeder et al. (2015) Lpg0393 Rab5, Rab21 and Rab22 GEF Activation of Rab5, Rab21 and Rab22 Sohn et al. (2015) Lpg1137 Syntaxin 17 Serine protease Blocks autophagosome formation Arasaki et al. (2017) LpnE (Lpg2222) OCRL1 and PI(3)P Unknown Recruits OCRL1 to LCV membranes and participates in LCV maturation Newton et al. (2007); Weber, Ragaz and Hilbi (2009) LpSpl Sphingosine Sphingosine-1 phosphate lyase Inhibits autophagy Rolando et al. (2016) LseA (Lpc2110) Syntaxin5, Syntaxin7, Syntaxin18, Vti1a, Vti1b, VAMP4, VAMP8 SNARE mimic Subverts vesicle trafficking King et al. (2015) LtpD (Lpw3701) (myo)-1- monophosphatase 1 (IMPA1) and PI(3)P Unknown Modulation of PI metabolism Harding et al. (2013) PieA (Lpg1963) Unknown Unknown Alters lysosome morphology Ninio, Celli and Roy (2009) PieE (Lpg1969) Rab1, Rab5, Rab6, Rab7 and Rab10 Unknown Modulation of Rab GTPase activity Mousnier et al. (2014) PlcC (CegC1/Lpg0012) PC, PG and PI Zinc metallophospholipase C Subverts host phospholipids Aurass et al. (2013) RalF (Lpg1950) Arf1 GEF Activates and recruits Arf1 to the LCV Nagai et al. (2002) RavZ (Lpg1683) LC3/Atg8 and PI(3)P Cysteine protease Deconjugates LC3/Atg8 from PE, inhibits autophagy Choy et al. (2012); Horenkamp et al. (2015) RidL (Ceg28/Lpg2311) Vps29 and PI(3)P Unknown Inhibits retrograde trafficking Finsel et al. (2013) SdcA (Lpg2510) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Luo et al. (2015) SdeA (Lpg2157) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeB (Lpg2156) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdeC (Lpg2153) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SdhA (Lpg0376) Unknown Unknown Maintains LCV integrity Creasey and Isberg (2012) SetA (Lpg1978) PI(3)P UDP-glucosyltransferase Subversion of vesicle trafficking Heidtman et al. (2009); Jank et al. (2012) SidC (Lpg2511/Llo3098) PI(4)P E3 ubiquitin ligase Participates in ER recruitment to the LCV Ragaz et al. (2008); Horenkamp et al. (2014); Hsu et al. (2014) SidD (Lpg2465) Rab1 and Rab35 De-AMPylase Modulation of Rab1 and Rab35 activity Neunuebel et al. (2011); Tan and Luo (2011) SidE (Lpg0234) Rab1, Rab6A, Rab30, Rab33B and RTN4 Ubiquitin ligase, phosphodiesterase, deubiquitylase Modulates ubiquitin dynamics on the LCV, recruits ER markers to the LCV Bhogaraju et al. (2016); Qiu et al. (2016); Kotewicz et al. (2017) SidF (Lpg2584) PI(3,4)P2 and PI(3,4,5)P3 PI 3-phosphatase Modulation of PI metabolism Hsu et al. (2012) SidJ (Lpg2155) Rab33 Deubiquitinase Modulates ubiquitin dynamics on the LCV and participates in ER recruitment to the LCV Liu and Luo (2007); Qiu et al. (2017) SidK (Lpg0968) VatA Unknown Inhibits vacuolar H+-ATPase and LCV acidification Xu et al. (2010); Zhao et al. (2017) SidM (DrrA/Lpg2464) Rab1, Rab35 and PI(4)P GEF, AMPylase Modulation of Rab1 and Rab35 activity Machner and Isberg (2006); Murata et al. (2006); Brombacher et al. (2009); Müller et al. (2010) SidP (Lpg0130) PI(3)P and PI(3,5)P2 PI 3-phosphatase Modulation of PI metabolism Toulabi et al. (2013) VipD (Lpg2831) Rab5, Rab22, PE, PC and PI(3)P Rab5/Rab22-activated phospholipase A1 Subverts host phospholipids, blocks endosomal trafficking Ku et al. (2012); Gaspar and Machner (2014) Mycobacterium Ndk Rac1, Rab5 and Rab7 Nucleoside diphosphate kinase, GAP Blocks phagosomal maturation Sun et al. (2010, 2013) SapM PI(3)P and Rab7 PI(3)P 3-phosphatase Inhibits phagosomal maturation and autophagosome-lysosome fusion Vergne et al. (2005); Hu et al. (2015) MptpB PI(3)P PI(3)P 3-phosphatase Inhibits phagosomal maturation Beresford et al. (2007) Salmonella GtgE Rab29, Rab32 and Rab38 Protease Removes Rab GTPases from SCV Spanò, Liu and Galán (2011); Spano and Galan (2012); Spanò et al. (2016) PipB Unknown Unknown Localises to SCV and SIF Knodler et al. (2003) PipB2 Kinesin-1 Unknown Recruits kinesin-1 on SCV, participates in SIF extension, reorganises late endosome/lysosome compartments Henry et al. (2006) SifA SKIP, RhoA and PLEKHM1 Unknown Promotes SIF biogenesis, maintains vacuolar membrane integrity, enables continuous fusion of host vesicles to SCV membrane Boucrot et al. (2005); Ohlson et al. (2008); McEwan et al. (2015) SseF ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region Deiwick et al. (2006); Yu, Liu and Holden (2016) SseG ACBD3 (GCP60) Unknown Promotes SIF formation and SCV positioning at peri-Golgi region (Deiwick et al. (2006); Yu, Liu and Holden (2016) SseJ RhoA, RhoC, phospholipids and cholesterol Deacylase, acyltransferase Esterifies cholesterol in infected cells, regulates SCV membrane dynamics and inhibits SIF biogenesis LaRock et al. (2012); Kolodziejek and Miller (2015); Raines et al. (2017) SopB PI(3,5)P2, PI(3,4,5)P3 PI(4,5)P2 Pleiotropic PI polyphosphatase Modulation of PI metabolism Hernandez et al. (2004); Mallo et al. (2008) SopD2 Rab7, Rab8, Rab10, Rab32 and Rab34 GAP Antagonises SifA in regulation of membrane dynamics and SIF biogenesis, inhibits host endocytic trafficking D’Costa et al. (2015); Spanò et al. (2016); Teo et al. (2017) SpvB G-actin ADP-ribosyl transferase Inhibits actin polymerisation and autophagosome formation, downregulates SIF biogenesis Tezcan-Merdol et al. (2001); Chu et al. (2016) SteA PI(4)P Unknown Controls SCV membrane dynamics Domingues, Holden and Mota (2014); Domingues et al. (2016) View Large LOCATION, LOCATION, LOCATION: WHERE TO BUILD A COMFORTABLE HOME As mentioned above, a vacuolar lifestyle protects bacteria from the host innate immune cytosolic surveillance. On the other hand, material internalised within endosomes and vacuoles is destined for degradation along the endocytic pathway, and the cytosol is certainly more abundant in nutrients as compared to membrane-bound compartments. Thus, a successful vacuolar lifestyle requires the evolution of strategies to deviate bacteria-containing vacuoles from the lysosomal route and to converge nutrients to the newly established replicative niche. Escaping degradation is achieved by two main strategies: either by arresting vacuole maturation at several stages between endocytic vesicles and lysosomes (Via et al.1997; Beron et al.2002; Mukherjee et al.2002; Rikihisa 2017) or by contacting host cell organelles such as the ER and the Golgi apparatus to generate unique, hybrid compartments (Hackstadt et al.1996; Tilney et al.2001) (Fig. 2). It is remarkable to note that either strategy requires extensive reprogramming of eukaryotic cell membrane trafficking and signalling pathways, which is coordinated by spatially isolated pathogens through vacuolar membranes. Vacuolar pathogens replicating within arrested endocytic vacuoles can either block endocytic maturation very early, therefore generating replicative niches with neutral pH (this is the case for M. tuberculosis and Ehrlichia chaffeensis for example), or delay their manipulation of membrane trafficking until later time points of infection, exposing themselves to acidic environments (a strategy adopted by S. enterica, C. burnetii and Brucella) (Fig. 2). Microbes belonging to the second group have adapted to acidic (and degradative in the case of C. burnetii) environments to the point that metabolism and/or translocation of effector proteins require the acidification of the replicative niche (Fig. 2). Figure 2. View largeDownload slide ‘Membrane code’ of bacteria-containing vacuoles. Early translocation of effector proteins allows L. pneumophila to drive the escape from the endocytic maturation and generate LCVs with neutral pH. This is achieved by manipulating the lipid signature of LCVs, which become mainly enriched in PI and PI(4)P, and by intersecting the early secretory pathway at ER exit sites (ERES). Thus, LCVs are decorated by ER membranes and are positive for typical secretory Rab GTPases such as Rab1, 14 and 8 (blue circles). The recruitment of a large number of Rab GTPases regulating different membrane trafficking pathways at Chlamydia inclusions is indicative of the complex interactions that this bacterium establishes with the infected cell. Transport to the MTOC facilitates interactions with the Golgi apparatus which is exploited as a source of membranes and lipids by Chlamydia. Mycobacterium uses translocated effector proteins to deplete PI(3)P from MCVs thereby perturbing the recruitment of Rab GTPases involved in membrane remodelling along the endocytic maturation pathway. As a result, MCVs are characterised by neutral pH and early endocytic (EE) Rab GTPases. Membrane damage by the bacterial secretion system may lead to membrane rupture and cytoplasmic escape of Mycobacteria. Other vacuolar pathogens require acidification of their replicative environment to activate metabolism and/or translocation of effector proteins. Salmonella SPI-2 effectors are translocated upon interactions of SCVs with late endosomal (LE) compartments and these are involved in the detoxification of SCVs to allow bacterial replication. SCVs are thus positive for early and late endocytic Rab GTPases including Rab5, 7, 11 and 14. Rab2 is also observed at SCVs and is indicative of fusion events between SCVs and COPII-positive secretory vesicles which facilitate SCVs rupture and cytoplasmic dissemination of Salmonella. Salmonella Typhimurium prevents Rab32 recruitment at SCVs, which has been demonstrated to regulate the species specificity of this pathogen. Coxiella burnetii metabolism and effector protein translocation is activated upon fusion of CCVs with lysosomes (Ly). Effector proteins co-opt the autophagy (Au) pathway, which is involved in homotypic fusion events between CCVs. Autophagy regulation is mediated by the manipulation of PI metabolism at the surface of CCVs which become enriched in PI(3)P. Thus, CCVs are positive for early and late Rab GTPases (Rab5 and 7, respectively) and also in the autophagy-related Rab24. Early Brucella-containing vacuoles (eBCVs) follow the endocytic maturation pathway until acidification of vacuolar pH upon fusion with lysosomes and the acquisition of the late endosomal Rab7. This triggers the translocation of Brucella effectors, which mediate the detoxification of BCVs by intersecting the early secretory pathway at ERES and COG complexes. Contacts with ER membranes and the acquisition of the ER-associated Rab2 drive the transformation into replicative BCVs (rBCVs) that support bacterial replication. Later during infection, rBCVs intersect the autophagy pathway and transform into autophagic BCVs (aBCVs), thus facilitating release from infected cells into the extracellular environment. Figure 2. View largeDownload slide ‘Membrane code’ of bacteria-containing vacuoles. Early translocation of effector proteins allows L. pneumophila to drive the escape from the endocytic maturation and generate LCVs with neutral pH. This is achieved by manipulating the lipid signature of LCVs, which become mainly enriched in PI and PI(4)P, and by intersecting the early secretory pathway at ER exit sites (ERES). Thus, LCVs are decorated by ER membranes and are positive for typical secretory Rab GTPases such as Rab1, 14 and 8 (blue circles). The recruitment of a large number of Rab GTPases regulating different membrane trafficking pathways at Chlamydia inclusions is indicative of the complex interactions that this bacterium establishes with the infected cell. Transport to the MTOC facilitates interactions with the Golgi apparatus which is exploited as a source of membranes and lipids by Chlamydia. Mycobacterium uses translocated effector proteins to deplete PI(3)P from MCVs thereby perturbing the recruitment of Rab GTPases involved in membrane remodelling along the endocytic maturation pathway. As a result, MCVs are characterised by neutral pH and early endocytic (EE) Rab GTPases. Membrane damage by the bacterial secretion system may lead to membrane rupture and cytoplasmic escape of Mycobacteria. Other vacuolar pathogens require acidification of their replicative environment to activate metabolism and/or translocation of effector proteins. Salmonella SPI-2 effectors are translocated upon interactions of SCVs with late endosomal (LE) compartments and these are involved in the detoxification of SCVs to allow bacterial replication. SCVs are thus positive for early and late endocytic Rab GTPases including Rab5, 7, 11 and 14. Rab2 is also observed at SCVs and is indicative of fusion events between SCVs and COPII-positive secretory vesicles which facilitate SCVs rupture and cytoplasmic dissemination of Salmonella. Salmonella Typhimurium prevents Rab32 recruitment at SCVs, which has been demonstrated to regulate the species specificity of this pathogen. Coxiella burnetii metabolism and effector protein translocation is activated upon fusion of CCVs with lysosomes (Ly). Effector proteins co-opt the autophagy (Au) pathway, which is involved in homotypic fusion events between CCVs. Autophagy regulation is mediated by the manipulation of PI metabolism at the surface of CCVs which become enriched in PI(3)P. Thus, CCVs are positive for early and late Rab GTPases (Rab5 and 7, respectively) and also in the autophagy-related Rab24. Early Brucella-containing vacuoles (eBCVs) follow the endocytic maturation pathway until acidification of vacuolar pH upon fusion with lysosomes and the acquisition of the late endosomal Rab7. This triggers the translocation of Brucella effectors, which mediate the detoxification of BCVs by intersecting the early secretory pathway at ERES and COG complexes. Contacts with ER membranes and the acquisition of the ER-associated Rab2 drive the transformation into replicative BCVs (rBCVs) that support bacterial replication. Later during infection, rBCVs intersect the autophagy pathway and transform into autophagic BCVs (aBCVs), thus facilitating release from infected cells into the extracellular environment. A remarkable example of how bacteria sense environmental pH to trigger virulence is provided by S. enterica. Upon invasion of host cells, SCVs follow the endocytic maturation pathway until merging with lysosomes (Mukherjee et al.2002). The acidification of SCVs is required for the assembly of the SPI-2 T3SS, but it is only upon insertion into the SCV membrane and sensing of the neutral pH of the host cell cytosol that effector translocation occurs (Yu et al.2010). Indeed, translocation is triggered by the dissociation and degradation of the T3SS core proteins SsaM, SpiC and SsaL, upon sensing of the cytosol neutral pH (Yu et al.2010). In order to prevent bacterial degradation by host cells, S. enterica uses SPI-2 effector proteins including SopD2 and SifA to detoxify lysosomes by subverting the activity of the small GTPases Rab7 and Rab9 (see below). Of note, recent proteomic analysis of isolated SCVs generated after 30 min and 3 h of infection highlighted an enrichment in ER-associated proteins, indicating that SCVs may also intercept the ER at nascent and intermediate stages of SCV formation (Santos et al.2015). It has been proposed that the observed membrane interactions between SCVs and the ER may provide an alternative SCV maturation pathway which resolves in the previously described vacuolar escape and cytosolic hyper-replication of Salmonella (Knodler et al.2010; Santos et al.2015) (Fig. 2). Coxiella burnetii offers another interesting example of how bacteria can adapt to harsh intracellular compartments. This microbe replicates within an acidic, degradative compartment with lysosomal features (Larson et al.2016). Differently from other pathogens following the endocytic pathway, which replicate in multiple, relatively small tight-fitting compartments, C. burnetii replicative vacuoles are extremely large and fusogenic, which leads to the generation of a single parasitophorous vacuole per infected cell (Larson et al.2016) (Fig. 2). Homotypic fusion of CCVs is facilitated by the autophagy-related SNARE protein syntaxin-17 and the C. burnetii effector protein CvpB/Cig2 (McDonough et al.2012; Newton et al.2014; Martinez et al.2016). CvpB-mediated manipulation of phosphoinositide (PI) metabolism illustrated below is likely at the basis of a subversion of the autophagy machinery to favour CCV biogenesis (Kohler et al.2016; Martinez et al.2016) (Figs 2 and 3). Emerging evidence indicates that the CCV intercept multiple membrane trafficking pathways as a mean to reroute membrane components for vacuole biogenesis (Larson et al.2013; Latomanski et al.2016; Justis et al.2017). Similarly to S. enterica, C. burnetii metabolism is also induced upon acidification of the CCV; however, the mechanisms behind the activation of C. burnetii metabolism by low pH remain to be elucidated. It is possible that the interactions with autophagosomes are required to deliver nutrients to the otherwise impermeable CCV (Heinzen et al.1996; Gutierrez et al.2005). In addition, proton gradients may facilitate the transport of metabolites and/or that gene transcription is under the regulation of pH sensors (Omsland et al.2008). Interestingly, vacuolar acidification is also required to trigger effector protein translocation by the C. burnetii Dot/Icm T4SS, as illustrated by chemically or genetically perturbing endosomal maturation (Newton, McDonough and Roy 2013). Of note, C. burnetii is the only vacuolar bacterium that is capable of surviving and replicating within a degradative compartment (Howe et al.2010). How C. burnetii resists to lysosomal degradation remains unclear; however, this does not require bacterial metabolism nor the translocation of effector proteins as chloramphenicol-treated bacteria and dot/icm mutants remain viable within lysosomes (Howe et al.2003; Beare et al.2011). Figure 3. View largeDownload slide ‘Friend or foe’, the diverse interactions between vacuolar pathogens and the autophagy machinery. All vacuolar pathogens discussed in this review interact with the autophagy machinery. Some of these interactions however benefit vacuole biogenesis and/or bacterial replication (blue background), whereas others are detrimental to the infectious process (red background). Francisella blocks ATG5-mediated autophagy by a yet unidentified effector protein. However, upon rupture of Francisella-containing vacuoles (FCVs), cytosolic bacteria can be captured by an ATG5-independent autophagy mechanism to generate a non-degradative replicative niche. Ehrlichia translocates the effector protein Etf-1, which interacts with Beclin1, Rab5 and Vps34 to recruit autophagosomes to the replicative niche. Similarly, Anaplasma translocates the effector protein Ats-1, which also interacts with Beclin-1 to recruit autophagosomes to Anaplasma-containing vacuoles. Coxiella translocates the effector protein CvpB/Cig2 which perturbs the activity of the PI3-kinase PIKfyve, thus favouring the autophagy-mediated homotypic fusion of Coxiella-containing vacuoles (CCVs). Also, autophagy is involved in membrane repair following CCVs damage. Upon maturation of Brucella-containing vacuoles (BCVs) to replicative BCVs (rBCVs) autophagy is recruited by an unknown effector protein for the generation of autophagic BCVs (aBCVs), which facilitate the transmission of the pathogen to bystander cells. Autophagy is also involved in membrane repair of Salmonella-containing-vacuoles (SCVs) following T3SS-mediated damage. Conversely, autophagy is also involved in clearance of ubiquitin-tagged (Ub) cytoplasmic Salmonella upon rupture of the SCV. Similarly, autophagosomes are recruited to Mycobacterium-containing vacuoles upon membrane damage induced by ESX-1. Vacuoles subsequently fuse with lysosomes, leading to bacterial degradation. Finally, Legionella translocates the effector proteins RavZ, Lpg1137 and LpSpl to block autophagy and the recruitment of autophagosomes to the Legionella-containing vacuole (LCV). Au, autophagosomes; Ly, lysosomes. Figure 3. View largeDownload slide ‘Friend or foe’, the diverse interactions between vacuolar pathogens and the autophagy machinery. All vacuolar pathogens discussed in this review interact with the autophagy machinery. Some of these interactions however benefit vacuole biogenesis and/or bacterial replication (blue background), whereas others are detrimental to the infectious process (red background). Francisella blocks ATG5-mediated autophagy by a yet unidentified effector protein. However, upon rupture of Francisella-containing vacuoles (FCVs), cytosolic bacteria can be captured by an ATG5-independent autophagy mechanism to generate a non-degradative replicative niche. Ehrlichia translocates the effector protein Etf-1, which interacts with Beclin1, Rab5 and Vps34 to recruit autophagosomes to the replicative niche. Similarly, Anaplasma translocates the effector protein Ats-1, which also interacts with Beclin-1 to recruit autophagosomes to Anaplasma-containing vacuoles. Coxiella translocates the effector protein CvpB/Cig2 which perturbs the activity of the PI3-kinase PIKfyve, thus favouring the autophagy-mediated homotypic fusion of Coxiella-containing vacuoles (CCVs). Also, autophagy is involved in membrane repair following CCVs damage. Upon maturation of Brucella-containing vacuoles (BCVs) to replicative BCVs (rBCVs) autophagy is recruited by an unknown effector protein for the generation of autophagic BCVs (aBCVs), which facilitate the transmission of the pathogen to bystander cells. Autophagy is also involved in membrane repair of Salmonella-containing-vacuoles (SCVs) following T3SS-mediated damage. Conversely, autophagy is also involved in clearance of ubiquitin-tagged (Ub) cytoplasmic Salmonella upon rupture of the SCV. Similarly, autophagosomes are recruited to Mycobacterium-containing vacuoles upon membrane damage induced by ESX-1. Vacuoles subsequently fuse with lysosomes, leading to bacterial degradation. Finally, Legionella translocates the effector proteins RavZ, Lpg1137 and LpSpl to block autophagy and the recruitment of autophagosomes to the Legionella-containing vacuole (LCV). Au, autophagosomes; Ly, lysosomes. Bacteria of the genus Brucella are characterised by an intracellular lifestyle that includes both maturation of BCVs along the endocytic pathway and fusion with host cell organelles (Pizarro-Cerda et al.1998; Celli et al.2003; Celli, Salcedo and Gorvel 2005; Starr et al.2008) (Fig. 2). Upon bacterial internalisation, BCVs interact with early and late endosomes, as indicated by the presence of typical markers of these vesicular compartments at the BCVs (Pizarro-Cerda et al.1998). Despite Brucella sensitivity to lysosomal degradation, BCVs also partially fuse with lysosomes as the expression of the virB T4SS operon is induced at low pH (Boschiroli et al.2002; Starr et al.2008). Translocation of effector proteins allows Brucella to do something remarkable, which is to switch gears from the endocytic pathway to the secretory pathway. Indeed, by a mechanism that remains to be fully elucidated, BCVs interact with ER exit sites (ERES) and acquire ER membranes (Celli, Salcedo and Gorvel 2005; Myeni et al.2013). Furthermore, it has been recently reported that Golgi-derived vesicles participate to BCVs biogenesis (Miller et al.2017) (Fig. 2). Surprisingly though, further maturation of BCVs also involves the subversion of the autophagy pathway as illustrated by the appearance of multi-layered replicative vacuoles that are positive for the autophagy marker LC3 (Starr et al.2012) (Fig. 2, 3). Inhibition of the autophagy machinery correlates with a reduced cell-to-cell transmission of Brucella, suggesting that subversion of autophagy is required at the latest stage of BCV maturation (Starr et al.2012). Accordingly, conditional expression of the VirB11 ATPase demonstrated a role of the Brucella T4SS in the transition from rBCVs to aBCVs and the following cell-to-cell transmission in bone marrow-derived macrophages (Smith et al.2016). Finally, infection of human trophoblasts with Brucella suis and B. abortus revealed alternative replicative niches for these bacteria. Indeed, these pathogens replicate in large, acidic inclusions that fail to acquire ER markers but remain positive for the lysosomal markers LAMP1 and CD63 (Salcedo et al.2013). Whether this represents an adaptation of Brucella to less permissive cells remain to be defined. In favour of this hypothesis, B. melitensis retains the ability of replicating within ER-positive BCVs in extravillous trophoblasts, suggesting that this species can overcome the restrictions imposed by these cells (Salcedo et al.2013). Interaction and fusion of bacteria-containing vacuoles with host cell organelles, which are typical of L. pneumophila and Chlamydia species, favours the biogenesis of replicative compartments characterised by neutral luminal pH. Legionella pneumophila uses a Dot/Icm T4SS apparatus to deliver an astonishing number of effector proteins into the cytosol of infected cells. These manipulate several host cell processes, mediating multiple interactions with the endosomal and the secretory membrane trafficking pathways, culminating with the association of LCVs with the ER (Horwitz 1983; Swanson and Isberg 1995; Robinson and Roy 2006) (Fig. 2). LCVs escape the endosomal maturation pathway early after bacterial internalisation, likely due to the activation of the Dot/Icm secretion system upon contacting the host plasma membrane during phagocytosis (Hubber and Roy 2010). Thus, LCVs acquire ER markers such as KDEL and calnexin (Lu and Clarke 2005) and are anchored to microtubules. LCVs interactions with the ER also occur at ERES and require the activity of host small GTPases including Rab1, Arf1 and Sar1 (Kagan and Roy 2002; Derré and Isberg 2004; Kagan et al.2004). ER-LCV contacts are mediated by the non-canonical pairing between plasma membrane-derived t-SNAREs (syntaxins, SNAP23) on LVCs (acquired upon phagocytosis) and ER v-SNARE Sec22b (Arasaki and Roy 2010). Legionella escapes autophagosomal degradation by translocating effector proteins with protease activity. Lpg1137 is a serine protease specifically targeting Syntaxin 17, leading to the inhibition of omegasome formation (Arasaki et al.2017); the cysteine protease RavZ irreversibly cleaves lipidated LC3 (Choy et al.2012) and LpSpl is a sphingosine-1 phosphate lyase that reduces sphingosine levels in infected cells, thus blocking autophagy (Rolando et al.2016) (Fig. 3). Moreover, LCVs are decorated with small GTPases of the Rab family (Fig. 2), Rap1 and Ran, many of which are implicated in intracellular replication of L. pneumophila (Urwyler et al.2008; Rothmeier et al.2013; Hoffmann et al.2014; Schmölders et al.2017). Recent reports illustrate the role of the host dynamin-like, large GTPase atlastin3 (Atl3) and reticulon 4 (Rtn4) in membrane fusion between the LCV and the ER, which is essential for vacuole expansion and bacterial replication (Kotewicz et al.2017; Steiner et al.2017). The Sde family of L. pneumophila effector proteins, and in particular SdeC, directly targets and ubiquitinates Rtn4 by a sequential ADP-ribosyltransferase and nucleotidase activity encoded by the bacterial effector, thus independently of the ubiquitination machinery of the host (Kotewicz et al.2017). Similarly to L. pneumophila, bacteria of the genus Chlamydia also use translocated effector proteins to escape the endocytic maturation pathway early after internalisation to generate a replicative niche called inclusion, which is characterised by a neutral pH (Heinzen et al.1996) (Fig. 2). In this case, however, the presence of the motor protein dynein at the inclusion membrane mediates the microtubule-driven relocation of the inclusion next to the microtubule organising centre (MTOC), possibly facilitating interactions with the Golgi apparatus (Grieshaber, Grieshaber and Hackstadt 2003). En route to the MTOC, the inclusion acquires a surprising number of Rab GTPases (see below), which is indicative of the complex network of interactions between the Chlamydia replicative niche and membrane trafficking pathways (Fig. 2). Interestingly, certain Rabs associate with the inclusion in a species-specific manner. Thus, Rab1, 4 and 11 are found on inclusions generated by all chlamydial species, whereas Rab6 and Rab10 associate with Chlamydia trachomatis and Ch. pneumoniae, respectively (Rzomp et al.2003). As mentioned above, the characterisation of the inclusion proteome identified 351 proteins and reported an enrichment in sorting nexins, cytosolic proteins capable of interacting with phospholipids and sensing membranes with high curvature. In eukaryotes, these proteins that take part in the retromer, a multiprotein complex recycling transmembrane receptors to the trans-Golgi network (Aeberhard et al.2015). Interestingly, RNAi-mediated depletion of SNX5 resulted in an increase of the infectious progeny, suggesting that the retromer might restrict bacterial replication (Aeberhard et al.2015). Homotypic fusion of Chlamydia inclusions requires the bacterial effector protein IncA and is promoted by microtubule-mediated membrane traffic (Hackstadt et al.1999; Richards, Knowlton and Grieshaber 2013). A hallmark feature of Chlamydia intracellular replication is the acquisition of host-derived lipids (sphingolipids, glycerosphingolipids and cholesterol), which are obtained by re-routing Golgi-derived vesicles to the Chlamydia inclusion (Scidmore, Fischer and Hackstadt 1996; Carabeo, Mead and Hackstadt 2003; Su et al.2004). Of note, the identification of glycerophospholipids, including phosphatidylcholine and phosphatidylinositol, integrated within bacterial membranes indicates that host-derived lipids are transferred from the membranes of the inclusion to Chlamydia (Hackstadt, Scidmore and Rockey 1995; Hackstadt et al.1996; Scidmore, Fischer and Hackstadt 1996; Wylie, Hatch and McClarty 1997). BUILDING BLOCKS: HOST PATHWAY SUBVERSION BY PATHOGENS One of the most effective ways of stalling endosomal maturation is to interfere with the way eukaryotic cells recognise and dispatch vesicles along the many membrane trafficking highways. The identity of most membrane-bound compartments is essentially encoded in their lipid and Rab GTPases composition, which defines what has been earlier referred to as the ‘membrane code’ (Jean and Kiger 2012). Together, PIs and Rab GTPases control the recruitment of host effector proteins coordinating membrane trafficking, fission and fusion events (Jean and Kiger 2012). PIs are key players in the regulation of membrane trafficking (De Matteis and Godi 2004). Their remarkable flexibility in signal transduction relies on the phosphoinositol ring, which can be reversibly phosphorylated at positions 3, 4 and 5, thus generating seven different PI species (De Matteis and Godi 2004). Specific kinases and phosphatases rapidly modify PIs, thereby controlling their distribution in space and time. Lipid metabolism allows the precise and local modulation of essential cellular processes including endocytosis and phagocytosis, membrane tethering, fusion and fission and autophagosome formation (De Matteis and Godi 2004). It is therefore not surprising that PIs and PIs metabolism are targeted by a growing number of cytosolic and vacuolar bacterial pathogens (Pizarro-Cerdá and Cossart 2004; Pizarro-Cerdá, Kühbacher and Cossart 2015). Bacterial effector proteins translocated by vacuolar pathogens can directly bind PIs for membrane targeting and anchoring. This is the case for the L. pneumophila effector proteins SidC, SidM, LidA, LpnE and RidL, which use PI(3)P and/or PI(4)P as anchors to localise at the surface of the LCV and favour interactions with the secretory pathway and the ER (Hilbi, Weber and Finsel 2011) (Fig. 2). Upon LCV localisation, RidL binds the Vps29 retromer subunit and displaces the Rab7 GTPase-activating protein (GAP) TBC1D5, thus blocking endosomes-to-Golgi traffic (Finsel et al.2013). SidM activates the small GTP-ase Rab1 (Machner and Isberg 2006) (see below); LidA interacts with ampylated Rab1, favouring interactions between LCVs and the ER (Machner and Isberg 2006); LpnE recruits the host PI 5-phosphatase OCRL to the Legionella replicative niche (Weber, Ragaz and Hilbi 2009). OCRL is also actively recruited at chlamydial inclusions by means of a yet unidentified Chlamydia effector protein (Moorhead et al.2010). In both cases, OCRL recruitment has been associated with the presence of PI(4)P at LCVs and inclusions (Weber, Ragaz and Hilbi 2009; Moorhead et al.2010). Interestingly, however, inhibition of OCRL has opposite effects on the intracellular fate of these two pathogens: Legionella growth is enhanced by OCRL depletion, suggesting that the phosphatase restricts bacterial growth, whereas optimal biogenesis of the chlamydial inclusion requires OCRL (Weber, Ragaz and Hilbi 2009; Moorhead et al.2010). Bacteria effector proteins can also manipulate PI metabolism either directly, by means of eukaryotic-like kinases and phosphatases secreted by the pathogen, or indirectly, by modulating the recruitment of host PI-metabolising enzymes (Pizarro-Cerdá and Cossart 2004). Collectively, manipulating the lipid profile of their host-derived replicative niche represents an effective method to modulate the interactions between bacteria-containing vacuoles and the endosomal maturation pathway and to ensure optimal intracellular replication (Fig. 2). Consequently, vacuolar pathogens that escape the endocytic maturation pathway early after internalisation tend to exclude PIs that facilitate the intersections with this pathway (Fig. 2). For example, M. tuberculosis depletes its replicative niche of PI(3)P, in order to block phagosomal maturation and avoid fusion with degradative compartments. This is achieved via the PI analogue mannose-capped lipoarabinomannan (ManLAM), which blocks the activity of the type III PI 3-kinase Vps34 (Fratti et al.2003), the secretion of the PI(3)P phosphatase SapM (Vergne et al.2005) and of the broader range phosphatase MptpB (Beresford et al.2007). Reducing the levels of PI(3)P impairs the recruitment of PI(3)P-binding fusion-promoting molecules such as EEA1 or Hrs, which are in turn essential for phagosome maturation (Fratti et al.2003; Vieira et al.2004). Similarly, L. pneumophila reduces the levels of PI(3)P at the LCV to escape the endocytic maturation pathway. This is achieved by means of the secreted effector protein SidP, which hydrolyses PI(3)P and PI(3,5)P2 (Toulabi et al.2013). In addition, the translocated effector protein SidF acts as a PI phosphatase whose activity leads to the accumulation of PI(4)P at the LCV (Hsu et al.2012) (Fig. 2). On the contrary, vacuolar pathogens that follow the endocytic pathway seeking the acidification of their replicative niche tend to facilitate the accumulation of PI(3)P at vacuolar membranes. To this aim, S. Typhimurium uses the SPI-1 substrate SopB, a pleiotropic PI polyphosphatase (Hernandez et al.2004; Mallo et al.2008). Besides promoting Salmonella uptake by acting at the plasma membrane during the early stages of infection, SopB also maintains high levels of PI(3)P and induces Rab5 recruitment to the surface of nascent SCVs, ultimately leading to the formation of large SCVs (Hernandez et al.2004; Mallo et al.2008) (Fig. 2). PI(3)P-binding effector sorting nexin-1 (SNX1) localises to SCVs in a SopB-dependent manner, promoting their remodelling and fostering communication with late endosomal compartments that characterise the later stages of SCV development (Bujny et al.2008). Along the same lines, the C. burnetii effector CvpB binds PI(3)P, thus perturbing the activity of PIKfyve, a PI 3-kinase which plays a key role in early endosomes maturation to late endosomes by phosphorylating PI(3)P to PI(3,5)P2 (Martinez et al.2016). PIKfyve inhibition enriches PI(3)P at CCVs (Martinez et al.2016) and sustains the autophagy pathway (Martin et al.2013), which is required for the homotypic fusion of CCVs (McDonough et al.2012; Newton et al.2014) (Figs 2 and 3). Thus, vacuolar pathogens interfere with lipid metabolism to camouflage their replicative niche and sidetrack it from the lysosomal degradation pathway. It is important to notice that many of these PI-binding bacterial effector proteins have evolved specific PI-binding domains that share no homology with those found in eukaryotes. Together with PIs, Rab GTPases play an essential role in defining the identity of intracellular compartments (Zerial and McBride 2001; Jean and Kiger 2012). Differently from PIs, Rab GTPases cycle between cytosol and cellular membranes, where they stably insert into the outer leaflet upon prenylation (Gomes et al.2003). Similar to PI-mediated signalling however, Rabs act as molecular switches transducing signals at topologically defined locations (Zerial and McBride 2001). Signal transduction depends on the structural conformation of Rabs, which in turn is tightly regulated by their active GTP- or inactive GDP-bound form (Zerial and McBride 2001; Hutagalung and Novick 2011). Specific guanine nucleotide exchange factors (GEFs) and GTP-activating proteins (GAPs) tightly regulate Rabs activity by loading GTP or facilitating its hydrolysis (Zerial and McBride 2001), which is reminiscent of the activity of PI kinases and phosphatases in the regulation of PI signal transduction. If on one hand the topological distribution of PIs is linked to their phosphorylation/dephosphorylation cycle, the human genome encodes over 60 Rab proteins, regulating specific steps of vesicles and organelles dynamics (Hutagalung and Novick 2011). Finally, it is important to note that several steps of intracellular membrane transport are controlled by the coordinated activity of PIs and Rabs (Jean and Kiger 2012). In the context of infections, investigating the ‘Rab signature’ of bacteria-containing vacuoles provides essential information to trace back the biogenesis of bacterial replicative niches and identify the host factors required for their generation. To this aim, proteomic characterisation of intracellular replicative niches has been achieved by isolating Legionella vacuoles and Chlamydia inclusions by immunomagnetic separation and Salmonella vacuoles by cell fractionation (Urwyler et al.2008; Hoffmann et al.2014; Aeberhard et al.2015; Santos et al.2015; Schmölders et al.2017). Besides their biological interest, this information may be used to design host-targeted antimicrobials to interfere with intracellular replication of bacteria. Over 500 proteins have been identified from isolated LCVs, including known host and bacterial components but also Rab GTPases such as Rab1, Rab8 and Rab14 (Urwyler et al.2008) (Fig. 2). Legionella pneumophila directly manipulates Rab GTPases to escape lysosomal degradation early after internalisation. The effector protein SidM uses the pool of PI(4)P generated by the phosphatase activity of SidF as an anchor to localise at LCVs (Brombacher et al.2009). There it sequesters and activates Rab1, the main regulator of the early secretory pathway. Rab1 activation is triggered by a specific GEF activity of SidM, via a GEF domain that shares no homology with other eukaryotic GEFs (Machner and Isberg 2006). Other effector proteins actively manipulate Rab1: AnkX (phosphorylcholination), LepB (GAP), SidD (deAMPylation), Lem3 (dephosphorylcholination) and LidA (Rab1 recruitment on LCV) (Qiu and Luo 2017). Rab1 activation at LCVs is required for fusion events between LCVs and ER membranes, which are mediated by non-canonical interactions between plasma membrane tSNARE syntaxin 3 present on LCVs and the ER vSNARE Sec22b (Kagan et al.2004; Arasaki and Roy 2010). Thus, L. pneumophila triggers fusion events between two intracellular compartments that are topologically distinct outside infection. The activity of SidM is enhanced by another L. pneumophila effector, LidA, which binds a number of Rab GTPases including Rab1, Rab6a and Rab8a but does not present any GEF or GAP activity (Machner and Isberg 2006; Schoebel et al.2009; Mihai Gazdag et al.2013). Interaction of LidA with Rab10 and Rab27a suggests that the effector protein could play a role in the manipulation of the phospholipid content of the LCV and vesicular transport along the secretory pathway (Yu et al.2015). Finally, the effector protein LepB acts as a GAP for Rab1, thus promoting its removal from LCVs (Ingmundson et al.2007). Of note, these effector proteins might have multiple activities as LepB also exhibits GAP activity for Rab3a, Rab8a, Rab13 and Rab35 in vitro. However, whether these functions have in vivo relevance remains to be elucidated. Chlamydia inclusions are characterised by a remarkable number of Rab GTPases (including Rab1, 4, 6, 11, 14, 34, 39), regulating multiple intracellular membrane trafficking steps, which is indicative of the complex biogenesis of this compartment (Damiani, Gambarte Tudela and Capmany 2014) (Fig. 2). Of note, the Golgi-associated Rab14 is required for the delivery of Golgi-derived sphingolipids to the inclusion (Capmany and Damiani 2010). Despite the abundance of Rab proteins identified at chlamydial inclusions, it is important to note that Rab5 and 7, typical markers of the degradative pathway identified in the majority of other bacterial replicative niches, are excluded from these compartments (Rzomp et al.2003) (Fig. 2). Unfortunately, the bacterial factors involved in the regulation of these processes remain largely unidentified, which is mainly due to the difficulties in the genetic manipulation of Chlamydia. Proteomics analysis of SCVs validated the previously reported presence of the late endosomal marker Rab7 and the early endosomal marker Rab5 and revealed the presence of Rab2a, a key regulator of ER-to-Golgi membrane traffic, thus corroborating the observation of SCV/ER MCS at the ultrastructural level (Santos et al.2015). To date, the effector proteins responsible for driving SCV/ER interactions remain to be identified. Of note, an earlier study comparing model phagosomes with SCVs revealed an extended network of Rab GTPases involved in the biogenesis of the Salmonella replicative compartment (Smith et al.2007). Some Rabs were found to be excluded from SCVs, including Rab8, 13, 23, 32 and 35, whereas other Rabs including Rab5, 7, 11 and 14 are enriched at SCVs (Smith et al.2007) (Fig. 2). During vacuole biogenesis, Rab5 and its associated effectors are retained at the surface of the bacterial replicative niche, thus delaying the maturation of SCVs along the endocytic pathway. This is achieved via the combined activity of the SPI-1 effector proteins SopB and SopE (Mukherjee et al.2001; Hernandez et al.2004; Mallo et al.2008). The phosphatase activity of SopB depletes PI(3,5)P2, PI(3,4,5)P3 and PI(4,5)P2 from SCV membranes leading to the recruitment of the PI 3-kinase Vps34 and the generation of PI(3)P, the membrane anchor for Rab5 (Mallo et al.2008) (Fig. 2). Upon Rab5 recruitment at SCVs, the Rab5-specific GEF activity of SopE promotes fusion with early endosomes (Mukherjee et al.2001). In addition, upon activation of the SPI-2 secretion system by vacuolar acidification, the effector protein SopD2 is translocated in the cytosol of the host cell where it binds to and inhibits the activation of Rab7 (D’Costa et al.2015). This perturbs the recruitment of the Rab7 effector proteins RILP (Rab-interacting lysosomal protein) and FYCO1 (FYVE and coiled-coil domain-containing protein 1), thereby disrupting the regulation of microtubules-associated motor proteins, which are required for lysosomal biogenesis (D’Costa et al.2015). The above-mentioned exclusion of Rab32 from SCVs harbouring S. enterica serovar Typhimurium is of particular interest as it has been demonstrated that this Rab GTPase is responsible for host restriction of the typhoid fever agent S. enterica serovar Typhi (Spano and Galan 2012). Indeed, the SPI-1 effector protein GtgE, which is absent in S. Typhi, depletes Rab32 from SCVs by triggering the proteolytic cleavage of this small GTPase (Spano and Galan 2012) (Fig. 2). Rab32 controls traffic to lysosome-related organelles and it is likely involved in the delivery of antimicrobial factors to SCVs. Thus, proteolytic cleavage of Rab32 contributes to SCVs detoxification. Ectopic expression of gtgE in S. Typhi enables bacteria belonging to this serovar to deplete Rab32 from SCVs, thus extending their host range (Spano and Galan 2012). Accordingly, siRNA depletion of Rab32 from cells infected by S. Typhi allows bacteria to replicate in normally restrictive cells (Spano and Galan 2012). Finally, the SPI-2 effector protein SifA, which is also involved in the formation of SIFs, contributes to the detoxification of SCVs by forming a stable complex with the host proteins SKIP and Rab9, thereby perturbing the function of the motor protein kinesin, mannose-6-phosphate receptor trafficking and lysosomal function (McGourty et al.2012). Infections by M. tuberculosis provide another example of how bacteria can indirectly manipulate the Rab signature of their replicative niche by manipulating PI metabolism. As illustrated above, M. tuberculosis translocated effector proteins deplete bacteria-containing compartments of PI(3)P, which serves as an anchor for membrane targeting of the fusion-promoting proteins EEA1 and Hrs (Fratti et al.2003; Vieira et al.2004). These specifically bind PI(3)P via their FYVE domains and are required for the transition from early to late endosomes. Thus, MCVs are positive for the early and recycling endosomal markers such as Rab2, 5, 11, 14 and 22, indicative of the early arrest of vacuole maturation (Fig. 2). It has been reported that Rab14 plays a major role in arresting MCVs maturation as RNAi-mediated depletion of the GTPase or the expression of its dominant negative mutant result in the progression of MCVs along the degradative pathway. It is interesting to note that Rab14 seems to play a role in the biogenesis of bacterial replicative niches (Fig. 2): it mediates the delivery of host lipids to the Chlamydia inclusion (Capmany and Damiani 2010), localises at LCVs and restricts L. pneumophila replication (Hoffmann et al.2014), mediates the acidification of SCVs via the effector Nischarin (Kuijl et al.2013). In addition, Rab14 expression is potentially regulated by the C. burnetii effector protein CBU_1314 (Weber et al.2016a); however, a role for Rab14 in CCVs biogenesis remains to be investigated. How Brucella coordinates the recruitment of Rab GTPases at the vacuole remains largely unknown; however, it has been reported that the effector protein RicA interacts with Rab2, which controls ER-to-Golgi trafficking, thereby mediating its recruitment to BCVs (de Barsy et al.2011) (Fig. 2). Despite the higher affinity for GDP-bound Rab2, it seems unlikely that RicA exhibits a GEF activity for this small GTPase (de Barsy et al.2011). HOME DELIVERY: VACUOLAR LIFESTYLE AND ACCESS TO NUTRIENTS Intracellular pathogens rely on host cells for nutrient acquisition, the main sources being amino acids found in proteins, fatty acids found on lipid droplets and carbohydrates found on glycogen (Steele, Brunton and Kawula 2015). Nutritional requirements have been defined, at least in part, for a number of intracellular bacterial pathogens assessing growth defects associated with nutritional mutants (reviewed in Ray et al.2009). Alternatively, the development of an axenic culture medium for host cell-free growth of the obligate intracellular pathogen C. burnetii provides a beautiful example of how multiple approaches can be used to define the nutritional requirements of obligate intracellular pathogens (Omsland et al.2009). These include knowledge of the intracellular replicative niche, bacterial physiology and genome analysis. Systematic testing of buffer compositions coupled to robust assays allows to define bacteria metabolic activity, growth and infectivity (Omsland et al.2009; Omsland, Hackstadt and Heinzen 2013). If on one hand cytosolic bacteria have direct access to these resources, vacuolar pathogens have to develop strategies to import nutrients through vacuolar membranes. To this aim, targeting of host catabolic pathways is triggered by a number of pathogens. Lysosomes contain degradative enzymes that provide a source of amino acids necessary for the growth of intracellular pathogens such as Chlamydia (Ouellette et al.2011). Alternatively, Chlamydia is also capable of intercepting nutrient-rich Golgi-derived vesicles and multivesicular bodies from the recycling pathway (Bastidas et al.2013; Samanta et al.2017). Furthermore, Ch. trachomatis grows in a glycogen-rich vacuole (Gehre et al.2016). Glycogen is a multibranched polysaccharide used for energy storage in cells. Chlamydia is capable of acquiring this nutrient by two means: transport in bulk from the host cytosol and recruitment of the host UDP-glucose transporter SLC35D2 to the membrane of its inclusion followed by branching of monomeric UDP-glucose into glycogen by the bacterial T3-secreted glycogen synthase GlgA (Gehre et al.2016). The targeting of the autophagy machinery by several vacuolar pathogens, including Coxiella, Brucella, Anaplasma, Ehrlichia and Francisella, may also represent an important source of nutrients (Fig. 3). Coxiella vacuole size increases when autophagy is triggered by starvation in infected cells (Gutierrez et al.2005; Romano et al.2007). As illustrated above, recruitment of the autophagy machinery to the vacuole membrane is dependent on the effector protein CvpB/Cig2 (Newton et al.2014; Kohler et al.2016; Martinez et al.2016) (Fig. 3); however, while vacuole morphology is affected by mutations in cvpB/cig2, C. burnetii is still capable of replicating in these vacuoles, strongly suggesting that additional cellular mechanisms are hijacked by the bacterium to acquire nutrients. While C. burnetii vacuole remains acidic during infection, Anaplasma develops in a vacuole with early autophagosomal properties (Niu et al.2012). In this case, inhibition of autophagy suppresses the intracellular proliferation of Anaplasma, highlighting the importance of this catabolic pathway in the development of the bacterium (Niu et al.2012) (Fig. 3). Autophagy is important for another intracellular pathogen, E. chaffeensis. Even though this bacterium develops in a vacuole with early endosomal features, it requires a functional host autophagy machinery to proliferate. During the course of infection, E. chaffeensis secretes Etf-1, an effector protein that interacts with Rab5, Vps34 and Beclin 1 to induce the Rab5-regulated autophagy pathway (Lin et al.2016) (Fig. 3). Thus, host cell nutrients may be captured by Etf-1-induced autophagosomes and delivered to E. chaffeensis inclusions before their fusion with lysosomes (Lin et al.2016). Francisella tularensis evades xenophagy but increases the flux of ATG5-independent autophagy (Steele et al.2013) (Fig. 3). Of note, this is the only case where autophagy induction has been directly linked to nutrient acquisition by using radiolabelled amino acids (Steele et al.2013). Finally, Chlamydia, Coxiella, Anaplasma and Ehrlichia also manipulate host cholesterol trafficking pathways to access nutrient-rich compartments (Samanta et al.2017). Pathogens that cannot stand the degradative conditions encountered in lysosomes and autolysosomes developed alternative ways to acquire nutrients from the cell. For example, L. pneumophila boosts polyubiquitination of host cell proteins via AnkB and the eukaryotic SCF1 ubiquitin ligase complex (Price, Richards and Abu Kwaik 2014). This leads to increased proteasomal degradation of host cell proteins which generates a surplus of amino acids that can be transported to the LCVs and used by the bacterium (Price, Richards and Abu Kwaik 2014). Interestingly, L. pneumophila and F. tularensis trigger the upregulation of SLC1A5, a host cell amino acid transporter, which is important for intracellular replication of both pathogens (Wieland et al.2005; Barel et al.2012). If on one hand it has been proposed that SLC1A5 may insert into LCVs to facilitate the transport of amino acids from the cytosol to the vacuolar lumen in Legionella-infected cells, its role in Francisella infections remains to be defined. Finally, Legionella also uses glucose to fuel its central metabolism (Eylert et al.2010). Generation of free glucose in infected cells might be facilitated by the Legionella-secreted eukaryotic-like glucoamylase (GamA) which is capable of degrading glycogen and starch (Herrmann et al.2011). PLAYING HIDE AND SEEK: VACUOLE SENSING BY HOST CELLS As illustrated above, the biogenesis of intracellular replicative niches is a strategy adopted by a number of microbes to ‘hide in plain sight’ from cytosolic immune sensing. Interestingly however, this strategy exposes vacuolar pathogens to the host cell innate immune system. Three main mechanisms for the sensing of vacuolar pathogens have been described: (1) direct sensing of pathogen-derived ligands, (2) indirect sensing of pathogen through their effectors and (3) detection of vacuoles by the sensing of altered ‘self’ (Liehl, Zuzarte-Luis and Mota 2015). The direct sensing of pathogen-derived ligands is based on the direct interaction between immune receptors and pathogen ligands. A well-studied example is that of S. Typhimurium T3SS1 and T3SS2, which cause the activation of signals leading to the assembly of the inflammasome (Liehl, Zuzarte-Luis and Mota 2015), mediated by Toll-like and NOD-like innate immune receptors (TLRs and NLRs, respectively) (Broz, Ohlson and Monack 2012). Upon infection of macrophages, S. Typhimurium lipopolysaccharide (LPS) can be detected by TLR4, leading to proinflammatory cytokines production including interleukin-1β (IL-1β) (Broz, Ohlson and Monack 2012). After vacuole formation, NLR family apoptosis inhibitory proteins (NAIPs) detect effector proteins translocated by the T3SS1 into the host cytosol and trigger the activation of NRLs receptors such as NLRC4 (NOD-LRR-and CARD-containing 4) (Liehl, Zuzarte-Luis and Mota 2015). In turn, these signals will trigger the assembly of the inflammasome that activates the caspase-1 signalling cascade, resulting in the release of cytokines and the pathogen clearance by pyroptosis (Moltke et al.2013). Mice express multiple NAIPs, with NAIP1 and 2 recognising T3SS components and NAIP5 and 6 responding to flagellar components (Miao et al.2006). Humans, however, encode a single functional NAIP with broad specificity (Kortmann, Brubaker and Monack 2015; Reyes Ruiz et al.2017). NLRC4 can also work with NAIPs and together detect the T3SS (Zhao et al.2011). Elimination of S. Typhimurium by pyroptosis has in vivo relevance as orogastrically infected mice deficient for caspase-1 and NLRC4 have a larger load of bacteria in liver, spleen and mesenteric lymph nodes compared to the wild-type mice (Broz, Ohlson and Monack 2012). Pathogen recognition can also be facilitated by transporters embedded in the host-cell-derived membranes of bacteria replicative niches that mediate the delivery of pathogen-derived ligands across the vacuole. Two mechanisms have been described in Salmonella species: NOD2 receptor is activated upon binding to muramyl dipeptide, a peptidoglycan motif translocated across the vacuolar membrane by two host endolysomal transporters, SLC15A3 et SLC15A4 (Nakamura et al.2014). NOD2 activation induces the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signalling pathway, resulting in proinflammatory cytokines production and autophagy (Nakamura et al.2014). Alternatively, it has been suggested that the host IFN-induced p65 guanylate-binding proteins (GBPs), belonging to the dynamin family, can destabilise vacuolar membranes, thus exposing the pathogen to host immune recognition (Meunier et al.2014). Indeed, GBPs expression is triggered upon extracellular detection of Salmonella by TLR4 and it has been shown that more bacteria are released to the cytosol of wild-type infected cells as compared to GBP-deficient cells, suggesting indeed that GBPs contribute to destabilisation of the vacuolar membrane (Meunier et al.2014). GBPs are also involved in cytosolic detection of vacuolar bacteria leading to inflammasome activation by the caspase-11-dependent pathway. Thus, GBPs cause damages to SCV therefore releasing the bacteria to the cytosol where these will be recognised by autophagic receptors and destined to elimination. Moreover, besides extracellular recognition mediated by TLR4, bacterial LPS can be recognised in the cytosol by caspase-11 (Kayagaki et al.2013; Shi et al.2014) leading to inflammasome activation. Recent studies have demonstrated that bacterial outer mem