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Stress adaptation in a pathogenic fungus

Stress adaptation in a pathogenic fungus © 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 144-155 doi:10.1242/jeb.088930 REVIEW Alistair J. P. Brown*, Susan Budge, Despoina Kaloriti, Anna Tillmann, Mette D. Jacobsen, Zhikang Yin, ‡ § ¶ Iuliana V. Ene , Iryna Bohovych , Doblin Sandai , Stavroula Kastora, Joanna Potrykus, Elizabeth R. Ballou, Delma S. Childers, Shahida Shahana and Michelle D. Leach** ABSTRACT normally exists as a harmless commensal organism in the microflora of the skin, oral cavity, and gastrointestinal and urogenital tracts of Candida albicans is a major fungal pathogen of humans. This yeast most healthy individuals (Odds, 1988; Calderone, 2002; Calderone is carried by many individuals as a harmless commensal, but when and Clancy, 2012). However, C. albicans frequently causes oral and immune defences are perturbed it causes mucosal infections vaginal infections (thrush) when the microflora is disturbed by (thrush). Additionally, when the immune system becomes severely antibiotic usage or when immune defences are perturbed, for compromised, C. albicans often causes life-threatening systemic example in HIV patients (Sobel, 2007; Revankar and Sobel, 2012). infections. A battery of virulence factors and fitness attributes promote In individuals whose immune systems are severely compromised the pathogenicity of C. albicans. Fitness attributes include robust (such as neutropenic patients undergoing chemotherapy or transplant responses to local environmental stresses, the inactivation of which surgery), the fungus can survive in the bloodstream, leading to the attenuates virulence. Stress signalling pathways in C. albicans colonisation of internal organs such as the kidney, liver, spleen and include evolutionarily conserved modules. However, there has been brain (Pfaller and Diekema, 2007; Calderone and Clancy, 2012). rewiring of some stress regulatory circuitry such that the roles of a Candida is the fourth most common cause of hospital-acquired number of regulators in C. albicans have diverged relative to the bloodstream infections, over half of which can be fatal in some benign model yeasts Saccharomyces cerevisiae and patient groups (Perlroth et al., 2007). This high morbidity exists Schizosaccharomyces pombe. This reflects the specific evolution of despite the availability of specialised antifungal drugs such as the C. albicans as an opportunistic pathogen obligately associated with azoles, polyenes and echinocandins (Odds et al., 2003a; Brown et warm-blooded animals, compared with other yeasts that are found al., 2012b), reflecting the challenges in diagnosing systemic fungal across diverse environmental niches. Our understanding of C. infections, the resultant delays in treatment, and the limited choice albicans stress signalling is based primarily on the in vitro responses of effective antifungal drugs (Pfaller and Diekema, 2010; Brown et of glucose-grown cells to individual stresses. However, in vivo this al., 2012b). From the fungal perspective, it is clear that C. albicans pathogen occupies complex and dynamic host niches characterised can adapt effectively to diverse host niches. by alternative carbon sources and simultaneous exposure to The evolutionary history of C. albicans has established both its combinations of stresses (rather than individual stresses). It has pathogenic behaviour and also its properties as an experimental become apparent that changes in carbon source strongly influence system. Candida albicans is a member of the ascomycete phylum, stress resistance, and that some combinatorial stresses exert non- which includes the model yeasts Saccharomyces cerevisiae and additive effects upon C. albicans. These effects, which are relevant Schizosaccharomyces pombe. These benign model yeasts provide to fungus–host interactions during disease progression, are mediated paradigms against which C. albicans is often compared (Berman by multiple mechanisms that include signalling and chemical and Sudbery, 2002; Enjalbert et al., 2006; Noble and Johnson, crosstalk, stress pathway interference and a biological transistor. 2007). However, in evolutionary terms C. albicans is only distantly KEY WORDS: Candida albicans, Fungal pathogenicity, Heat shock, related to S. cerevisiae (circa 150 million years) and S. pombe (>400 Oxidative stress, Nitrosative stress, Osmotic stress, Cationic million years) (Galagan et al., 2005), and the latter evolutionary stress, Stress adaptation, Carbon metabolism distance represents greater separation than exists between humans and sharks. Furthermore, although ascomycetes are generally Introduction: Candida albicans – an opportunistic pathogen defined by their packaging of sexual spores into an ascus structure, of humans C. albicans has not been observed to undergo meiosis to generate Candida albicans is a major fungal pathogen of humans that spores. Rather, this diploid yeast, which until very recently was occupies a wide range of divergent niches within the host. It thought to be constitutively diploid (Hickman et al., 2013), displays a complex parasexual cycle. Candida albicans must undergo homozygosis at the mating type locus (MTL) and then undergo an School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. epigenetic switch to mating competent cells (the opaque form) Present address: Department of Molecular Microbiology and Immunology, Brown before it mates to form tetraploids (Noble and Johnson, 2007). This University, Providence, RI 02912, USA. Present address: Nebraska Redox is followed by chromosome loss to return to the diploid state Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588-0662, USA. Present address: Institut Perubatan and Pergigian Termaju, Universiti Sains (Forche et al., 2008). While parasexual recombination could have Malaysia, Pulau Pinang, Malaysia. **Present address: Department of Molecular contributed to the recent evolution of C. albicans, the population Genetics, University of Toronto, Medical Sciences Building, Toronto, Canada, structure is predominantly clonal (Cowen et al., 2002; Odds et al., M5S 1A8. 2007). Indeed, its recent evolution appears to have been driven *Author for correspondence ([email protected]) largely by its clonal behaviour as a pathogen. Candida albicans has not been associated with any particular environmental niche and This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted hence is thought to be obligately associated with warm-blooded use, distribution and reproduction in any medium provided that the original work is properly attributed. animals (Odds, 1988). Therefore, it is not surprising that this fungus The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 the host. For example, oxidative, nitrosative and heat shock List of abbreviations functions are induced when cells are phagocytosed by macrophages GFP green fluorescent protein or neutrophils (Rubin-Bejerano et al., 2003; Lorenz et al., 2004; HSE heat shock element Fradin et al., 2005), and the niche-specific induction of specific HSP heat shock protein stress responses has been confirmed by single cell profiling using MAPK mitogen-activated protein kinase diagnostic green fluorescent protein (GFP) fusions (Enjalbert et al., MAPKK MAP kinase kinase MAPKKK MAP kinase kinase kinase 2007; Miramón et al., 2012). Second, the virulence of C. albicans RCS reactive chlorine species in mouse models of infection is attenuated by the inactivation of key RNS reactive nitrogen species stress functions such as the stress-activated protein kinase (SAPK) ROS reactive oxygen species Hog1, the catalase Cat1 or the superoxide dismutase Sod1 (Wysong SAPK stress-activated protein kinase et al., 1998; Alonso-Monge et al., 1999; Hwang et al., 2002; Cheetham et al., 2011). Significant progress has been made in the has undergone the rapid evolution of virulence factors and fitness elaboration of stress-adaptive responses, their regulation in C. attributes associated with its pathogenicity (Butler et al., 2009; albicans and their divergence from the corresponding pathways in Nikolaou et al., 2009) as well as evolutionary rewiring of model yeasts. A brief overview of these mechanisms will be transcriptional and post-transcriptional circuitries relative to S. discussed here. This provides the platform for the main theme of this cerevisiae (Ihmels et al., 2005; Martchenko et al., 2007; Lavoie et review – stress adaptation in the context of complex and dynamic al., 2009; Baker et al., 2012; Sandai et al., 2012). These changes host niches (mentioned above), in which C. albicans cells must have had a significant impact on the evolution of stress adaptation respond to multiple environmental inputs, rather than to the in C. albicans (Brown et al., 2012a). individual stresses commonly studied in vitro. The loss of a bona fide sexual cycle has had a major impact on the experimental dissection of C. albicans pathobiology. Researchers Overview of stress adaptation mechanisms in C. albicans have had to rely mainly on genomic and molecular approaches, Stress signalling pathways are relatively well characterised in S. rather than genetic strategies to examine the virulence of this fungus cerevisiae and S. pombe. A number of the key regulators are (Noble and Johnson, 2007). Nevertheless, these approaches have evolutionarily conserved in C. albicans (Butler et al., 2009; revealed an armoury of virulence factors that promote the Nikolaou et al., 2009) (Fig. 1). However, the roles of some of these pathogenicity of C. albicans. Virulence factors have been defined as regulators have diverged (Enjalbert et al., 2003; Nicholls et al., those fungal factors that interact directly with host components 2004; Ramsdale et al., 2008; Cheetham et al., 2007), and C. albicans (Odds et al., 2003b). For example, reversible morphogenetic is relatively resistant to physiologically relevant stresses compared transitions between yeast, pseudohyphal and hyphal growth forms with model yeasts (Jamieson et al., 1996; Nikolaou et al., 2009). contribute to the virulence of C. albicans (Lo et al., 1997; Saville et This is consistent with the idea that stress responses in C. albicans al., 2003). Yeast forms are thought to promote dissemination, have been evolutionarily tuned to host niches. Stress signalling in C. whereas the filamentous forms are better suited to penetrate tissue. albicans has been described in a number of recent reviews (Chauhan Hyphae also display thigmotropic responses that appear to et al., 2006; Alonso-Monge et al., 2009b; Brown et al., 2009; Smith contribute to tissue penetration (Sherwood et al., 1992; Brand, et al., 2010; Brown et al., 2012a). Therefore, the purpose of this 2012). Initial colonisation is mediated by families of cell surface section is to summarise key stress signalling pathways, highlighting adhesins that promote adherence to host tissues (Staab et al., 1999; their relevance to infection. Hoyer et al., 2008). One of these adhesins, Als3, also acts as an invasin by promoting the invasion of endothelial cells (Phan et al., Heat shock 2007), contributing to the assimilation of the essential micronutrient The heat shock response is ubiquitous in nature. In eukaryotes, it iron (Almeida et al., 2008; Almeida et al., 2009) and to brain involves the induction of a defined set of heat shock proteins colonisation (Liu et al., 2011). Candida albicans expresses (HSPs), many of which promote the folding of client proteins or additional factors involved in iron and zinc assimilation, some of target aggregated or damaged proteins for degradation (Parsell and which are essential for virulence (Almeida et al., 2009; Citiulo et al., Lindquist, 1993; Feder and Hofmann, 1999). The response in C. 2012), and which are induced during renal infection (J.P. and albicans, as in other yeasts, is driven by the heat shock transcription A.J.P.B., unpublished). Candida albicans also secretes families of factor Hsf1 (Nicholls et al., 2009). Hsf1 is conserved from yeasts to hydrolytic enzymes including proteases, lipases and phospholipases humans and is essential for viability (Sorger and Pelham, 1988; (Naglik et al., 2003; Schaller et al., 2005) that enhance tissue Sarge et al., 1993; Wu, 1995). In response to acute heat shock, C. invasion, provide nutrients to support fungal growth and modulate albicans Hsf1 becomes phosphorylated and induces the expression host immune responses (Pietrella et al., 2010). These and other of target heat shock protein (HSP) genes via canonical heat shock factors are temporally and spatially regulated during colonisation elements (HSEs) in their promoters (Nicholls et al., 2009), an and disease progression, thereby enhancing C. albicans interaction that is conserved in other eukaryotes (Sorger and Pelham, pathogenicity. 1988; Jakobsen and Pelham, 1988; Holmberg et al., 2001). HSP Additional factors promote the virulence of C. albicans without gene induction leads to the refolding or degradation of damaged interacting directly with the host. These factors, which have been proteins, thereby promoting cellular adaptation to the thermal insult. termed ‘fitness attributes’ (Brown, 2005), include functions involved Indeed, in C. albicans heat shock induces polyubiquitin (UBI4) in metabolic and stress adaptation and act by tuning the expression, which is required for resistance to thermal stress (Roig physiological fitness of C. albicans cells to their local host and Gozalbo, 2003; Leach et al., 2011). The HSP90 gene is also microenvironment. Two main types of evidence have highlighted the activated in an Hsf1-dependent fashion (Nicholls et al., 2009). Heat importance of stress adaptation for the virulence of C. albicans. shock protein 90 (Hsp90) has been described as a molecular First, numerous genome-wide expression profiles have demonstrated transistor as it modulates the activity of client regulatory proteins that stress genes are induced when the fungus comes in contact with (Leach et al., 2012a). Following thermal adaptation, Hsp90 interacts The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Osmotic/ Enjalbert et al., 2006). This leads to the accumulation of glycerol, Thermal Nitrosative Oxidative cationic Cell wall damage stress stress stress the restoration of turgor pressure and the resumption of growth. stress Glycerol biosynthetic gene induction, glycerol accumulation and the successful adaptation of C. albicans cells to osmotic/cationic Ssk2 Bck1 Ste11 stresses are Hog1 dependent (San José et al., 1996; Smith et al., Pbs2 Mkk1 Hst7 2004). Hog1 Mck1 Cek1 Hog1 is a component of a highly conserved mitogen-activated Hsp90 protein (MAP) kinase pathway involved in osmo-adaptation in other Hsf1 Cta4 Cap1 Skn7 yeasts (Nikolaou et al., 2009; Smith et al., 2010). In C. albicans, this MAP kinase (MAPK) is activated by the MAP kinase kinase Glutaredoxin SODs & thioredoxin (MAPKK) Pbs2, which in turn is activated by a single MAP kinase catalase Glycerol Chaperones Yhb1 Cell wall remodelling systems accumulation kinase kinase (MAPKKK), Ssk2 (Arana et al., 2005; Cheetham et Glutathione Trehalose al., 2007) (Fig. 1). However, the upstream regulators that activate this MAPK module in response to osmotic stress have not been Stress adaptation established unambiguously in C. albicans. In S. cerevisiae, this Virulence MAPK module responds to two well-defined upstream branches (reviewed by Smith et al., 2010). The Sho1 branch activates Hog1 Fig. 1. Conserved stress regulators in Candida albicans. Evolutionarily signalling via Cdc42, Ste50, Ste20 and Cla4, and through the conserved mitogen-activated protein kinase (MAPK) signalling molecules MAPKKK Ste11 specifically in response to heat or osmotic stress. (red) and transcription factors (blue) contribute to the regulation of stress The Sln1 phospho-relay system includes Ypd1 and Ssk1, and functions in C. albicans (see ‘Overview of stress adaptation mechanisms in activates Hog1 signalling via the MAPKKKs Ssk2 and Ssk22 in C. albicans’). Hsf1 and Hsp90 operate in an autoregulatory circuit, whereby response to a broad range of stresses, including osmotic stress. synthesis of the biological transistor Hsp90 (green) is activated by Hsf1 in Candida albicans has orthologues for many of these proteins response to heat shock, and Hsp90 then downregulates Hsf1 (see ‘Overview of stress adaptation mechanisms in C. albicans’). These pathways are (Nikolaou et al., 2009), as well as proteins that are related to represented as linear pathways (for simplicity), but most probably operate in histidine kinases in S. cerevisiae and S. pombe (C. albicans Sln1, an integrated network. Heat shock pathway: Hsp90, heat shock protein 90; Chk1, Nik1) (Kruppa and Calderone, 2006). However, in C. Hsf1, heat shock transcription factor. Nitrosative stress pathway: Cta4, zinc albicans none of these histidine kinases or Ssk1 is essential for the cluster transcription factor; Yhb1, nitric oxide dioxygenase. Oxidative stress osmotic stress-induced activation of Hog1 (Chauhan et al., 2003; pathway: Cap1, AP-1 bZIP transcription factor; Skn7; putative response Kruppa and Calderone, 2006), suggesting that the Sln1 branch does regulator; SODs, superoxide dismutases. Hog1 signalling pathway: Ssk2, not transduce osmotic stress signals to Hog1. Furthermore, a ypd1 MAPK kinase kinase (MAPKKK); Pbs2, MAPK kinase (MAPKK); Hog1, MAPK/stress-activated protein kinase (SAPK). Cell integrity pathway: Bck1, sho1 double mutation does not block osmotic stress signalling to MAPKKK; Mkk1, MAPKK; Mkc1, MAPK. Mating/invasive growth pathway: Hog1 in C. albicans (Román et al., 2005), indicating that the Sho1 Ste11, MAPKKK; Hst7, MAPKK; Cek1, MAPK. branch is not essential for osmotic stress signalling either. Therefore, it is not yet clear how osmotic stress signals are transduced to Hog1, and there appears to have been significant evolutionary rewiring of physically with Hsf1 to downregulate the heat shock response in C. the upstream regulators of this stress pathway. albicans (Leach et al., 2012b) (Fig. 1). The inactivation of Hog1 attenuates the virulence of C. albicans Significantly, while other conserved stress regulatory circuits have (Alonso-Monge et al., 1999; Cheetham et al., 2011). However, this undergone evolutionary rewiring (see below), heat shock regulation is not attributable simply to the loss of osmotic or cationic stress has been maintained in C. albicans (Nicholls et al., 2009) despite its adaptation because Hog1 has been shown to execute additional obligate association with warm-blooded animals (Odds, 1988). functions. Hog1 is required for adaptation to other stresses, Presumably the fungus occupies thermally buffered niches in the modulates cellular morphogenesis, influences metabolism and host and is generally sheltered from the acute heat shocks that are affects cell wall functionality (Alonso-Monge et al., 1999; Alonso- imposed in the laboratory. Interestingly, mutations that block the Monge et al., 2003; Alonso-Monge et al., 2009a; Smith et al., 2004; activation of the heat shock response attenuate the virulence of C. Eisman et al., 2006). Nevertheless, several observations suggest that albicans (Nicholls et al., 2011). Mathematical modelling of the osmotic and cationic stress adaptation play significant roles in dynamic regulation of Hsf1 during thermal adaptation has provided certain host niches. First, NaCl concentrations can approach −1 an answer to this conundrum (Leach et al., 2012c). The Hsf1–HSE 600 mmol l in the kidney and be high in the urine (Ohno et al., regulon appears to be activated even during slow thermal transitions 1997; Zhang et al., 2004). Second, C. albicans cells are exposed to such as those suffered by febrile patients. This explains why Hsf1 K fluxes following phagocytosis by host immune cells (Da Silva- activation is essential for the virulence of C. albicans (Nicholls et Santos et al., 2002; Fang, 2004). Third, mathematical modelling of al., 2011). Clearly, the Hsf1–HSE regulon is critical for the osmotic stress adaptation in S. cerevisiae has highlighted the role of maintenance of thermal homeostasis, not merely for adaptation to this regulatory apparatus in mediating cellular osmo-homeostasis acute heat shocks. and the maintenance of water balance (Klipp et al., 2005), in addition to its role in adaptation to the acute osmotic shocks that Osmotic and cationic stress experimentalists tend to impose in vitro. Hence, Hog1-mediated Exposure to NaCl or KCl imposes osmotic and cationic stress, osmotic adaptation is likely to be required in many host niches. which causes rapid water loss, a reduction in cell size and loss of turgor pressure (Kühn and Klipp, 2012). This triggers the Cell wall stress phosphorylation and nuclear accumulation of the SAPK Hog1, Antifungal drugs such as caspofungin and chemicals such as which in turn mediates the activation of target genes including those Calcofluor White and Congo Red are often used to exert stress upon encoding glycerol biosynthetic enzymes (Smith et al., 2004; the cell wall of C. albicans in vitro (Wiederhold et al., 2005; Eisman The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 et al., 2006; Walker et al., 2008; Leach et al., 2012a). Caspofungin attenuates the induction of these genes, rendering C. albicans and Congo Red interfere with β-glucan synthesis and assembly, sensitive to oxidative stress (Alarco and Raymond, 1999; Enjalbert whereas Calcofluor White perturbs chitin assembly. The cell wall et al., 2006). The redox status of Cap1, and hence oxidative stress changes that occur in response to these artificial insults presumably adaptation, is modulated by the redox regulator thioredoxin (Trx1) reflect normal cell wall homeostasis during growth and development (da Silva Dantas et al., 2010). in the wild, as well as cell wall remodelling events that occur in The Hog1 MAPK pathway also contributes to oxidative stress response to stresses encountered during host–fungus interactions. resistance in C. albicans (Fig. 1). Inactivation of Hog1 and key The Hog1 pathway contributes to cell wall functionality and upstream regulators confer oxidative stress sensitivity (Alonso- regulates chitin biosynthetic functions (Eisman et al., 2006; Munro Monge et al., 2003; Chauhan et al., 2003; Smith et al., 2004; Kruppa et al., 2007). Two additional MAPK pathways contribute to cell wall and Calderone, 2006; da Silva Dantas et al., 2010; Smith et al., stress resistance in C. albicans: the cell integrity pathway (defined 2010). Oxidative stress signals appear to be transduced to Hog1 via by the MAPK Mkc1) and a second pathway that was originally the histidine kinases (Sln1, Chk1, Nik1), the response regulator Ssk1 characterised on the basis of its involvement in yeast-hypha and the peroxiredoxin Tsa1 (Kruppa and Calderone, 2006; Smith et morphogenesis (defined by the MAPK Cek1) (Fig. 1). Both al., 2010). An additional response regulator (Crr1) contributes to pathways are evolutionarily conserved in other fungi (Román et al., oxidative stress resistance in C. albicans, but is not required for 2007). Hog1 activation in response to H O (Bruce et al., 2011). The 2 2 The cell integrity pathway includes a MAPKK module that downstream molecular mechanisms that underlie Hog1-mediated incorporates the MAPKKK Bck1, the MAPKK Mkk1 and the oxidative stress resistance remain an area of active research. The MAPK Mkc1 (Alonso-Monge et al., 2006). Mkc1 activation by cell nuclear accumulation of Cap1 is not dependent on Hog1, and most wall stress is mediated through protein kinase C (Pkc1) signalling oxidative stress-induced transcripts are induced in a Hog1- (Paravicini et al., 1996; Alonso-Monge et al., 2006). The disruption independent fashion (Enjalbert et al., 2006). of Mkc1 confers sensitivity to cell wall stresses and elevated Numerous observations indicate that C. albicans cells are exposed temperatures (Navarro-García et al., 1995). Mkc1 inactivation does to oxidative stress during infection and that oxidative stress not increase the sensitivity of C. albicans to killing by neutrophils adaptation is essential for pathogenicity. There has been or macrophages (Arana et al., 2007), but does attenuate the virulence evolutionary expansion of the SOD gene family in C. albicans, with of C. albicans (Diez-Orejas et al., 1997). this pathogen carrying six superoxide dismutase genes. Transcript The morphogenetic MAPK (Cek1) pathway includes the profiling experiments have demonstrated that oxidative stress genes MAPKKK Ste11, the MAPKK Hst7 and the MAPK Cek1 (Brown, are induced following exposure to host macrophages and neutrophils 2002; Alonso-Monge et al., 2006). Components of this MAPK (Rubin-Bejerano et al., 2003; Lorenz et al., 2004; Fradin et al., module are also involved in the C. albicans mating response (Chen 2005), and during mucosal infection (Zakikhany et al., 2007), but et al., 2002), but Cek2 acts as the MAPK under these conditions. are not activated to the same extent during tissue infection (Thewes The Cek1 pathway is activated via the cell surface sensor Msb2 in et al., 2007; Walker et al., 2009; Wilson et al., 2009). These response to cell wall damaging agents and mutations that affect cell expression patterns have been confirmed by single cell profiling of wall integrity (Román et al., 2009; Cantero and Ernst, 2011). C. albicans cells tagged with diagnostic GFP fusions to oxidative Inactivation of components on the Cek1 pathway inhibits stress genes (Enjalbert et al., 2007; Arana et al., 2007; Miramón et filamentous growth under certain conditions and confers sensitivity al., 2012). The inactivation of genes involved in ROS detoxification, to cell wall stresses (Leberer et al., 1996; Csank et al., 1998; Eisman such as superoxide dismutates and catalase, renders C. albicans cells et al., 2006; Cantero and Ernst, 2011). Candida albicans cek1 more sensitive to phagocytic killing and attenuates the virulence of mutants are not hypersensitive to macrophage or neutrophil killing, the fungus (Wysong et al., 1998; Hwang et al., 2002; Fradin et al., but do display attenuated virulence (Csank et al., 1998; Arana et al., 2005; Frohner et al., 2009). Similar phenotypes are also observed 2007). following the perturbation of oxidative stress regulators. Candida albicans cap1 and hog1 mutants are killed more effectively by Oxidative stress phagocytes (Fradin et al., 2005; Arana et al., 2007), and hog1 and Candida albicans is relatively resistant to reactive oxygen species trx1 mutants display attenuated virulence (Alonso-Monge et al., −1 (ROS), tolerating over 20 mmol l hydrogen peroxide (H O ) under 1999; da Silva Dantas et al., 2010; Cheetham et al., 2011). Taken 2 2 some conditions (Jamieson et al., 1996; Nikolaou et al., 2009; together, the data suggest that C. albicans exploits robust oxidative Rodaki et al., 2009). This resistance is dependent on the AP-1-like stress responses to protect itself from phagocytic killing, but these transcription factor Cap1, which is an orthologue of S. cerevisiae responses become less vital as the fungus develops systemic Yap1 and S. pombe Pap1 (Alarco and Raymond, 1999), and upon infections. the response regulator Skn7 (Singh et al., 2004) (Fig. 1). Cap1 contains redox-sensitive cysteine residues near its carboxy terminus Nitrosative stress that become oxidised following oxidative stress. This leads to the Exposure to reactive nitrogen species (RNS), for example nitric Hog1-independent nuclear accumulation of Cap1 and the activation oxide, causes molecular damage such as the S-nitrosylation of the of its target genes via Yap1-responsive elements (YRE) in their thiol groups of cysteines in proteins and glutathione. RNS exert promoters (Zhang et al., 2000; Enjalbert et al., 2006; Znaidi et al., static rather than cidal effects upon C. albicans (Kaloriti et al., 2009). Cap1 targets include genes involved in the detoxification of 2012). Candida albicans responds to nitrosative stress by activating oxidative stress (e.g. catalase and superoxide dismutase: CAT1 and a defined set of genes that includes oxidative stress functions such SOD1), glutathione synthesis (e.g. gamma-glutamylcysteine as catalase (Cat1), glutathione-conjugating and -modifying enzymes, synthetase: GCS1), redox homeostasis and oxidative damage repair and NADPH oxidoreductases and dehydrogenases (Hromatka et al., (e.g. glutathione reductase and thioredoxin: GLR1 and TRX1). 2005). In addition, YHB1 expression is strongly induced. YHB1 is Together, these functions detoxify ROS and mediate cellular one of three genes encoding flavohaemoglobin-related proteins in adaptation to stress. Consequently, the inactivation of Cap1 C. albicans: YHB1, YHB4 and YHB5 (Ullmann et al., 2004; The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Tillmann et al., 2011). Of these, only YHB1 is induced in response to the types and intensities of stresses that C. albicans encounters to nitric oxide, and this gene encodes the major nitric oxide during its interactions with the host. Third, these stress responses are dioxygenase responsible for nitric oxide detoxification (Ullmann et intimately linked to the virulence of this pathogen (Brown et al., al., 2004; Hromatka et al., 2005). Following RNS detoxification, 2007; Brown et al., 2012a; Román et al., 2007). redox homeostasis is restored and S-nitrosylated adducts are repaired, allowing C. albicans to resume growth (A.T. and A.J.P.B., Adaptation to sequential stresses unpublished). Almost without exception, all of the above studies on stress Little is known about the signalling pathways that mediate the adaptation in C. albicans have examined the responses of cells to nitrosative stress response in C. albicans, or in other yeasts for that individual stresses following growth on glucose. Yet, as described matter. However, it has been shown that the zinc finger transcription above, this pathogen inhabits diverse, complex and dynamic niches factor Cta4 is responsible for activating YHB1 expression in in the host. In these niches C. albicans will be exposed to multiple response to RNS (Chiranand et al., 2008), and the inactivation of stresses. At times these stresses may be imposed sequentially. At either CTA4 or YHB1 confers nitrosative stress sensitivity (Ullmann other times, multiple stresses are imposed simultaneously such that et al., 2004; Chiranand et al., 2008) (Fig. 1). the fungus is exposed to ‘combinatorial stress’. Furthermore, as In addition to ROS, the molecular armoury of phagocytic cells glucose is either limiting or absent from many host niches, C. includes RNS that contribute to fungal killing (Rementería et al., albicans cells must adapt to these stresses whilst exploiting 1995; Vázquez-Torres and Balish, 1997; Brown, 2011). Not alternative carbon sources. Recent data have revealed that these surprisingly, therefore, nitrosative stress genes are induced following factors significantly influence stress adaptation in C. albicans. This phagocytic attack (Fradin et al., 2005; Zakikhany et al., 2007). section addresses adaptation to sequential stresses, and the following Nitrosative stress genes are also upregulated during mucosal section discusses the impact of combinatorial inputs upon stress infections (Zakikhany et al., 2007). However, the response is not adaptation. strongly activated during systemic infection (Thewes et al., 2007; With regard to sequential stresses, it has been known for some Walker et al., 2009), and the inactivation of Yhb1 or Cta4 only time that prior exposure to a non-lethal dose of a stress can protect causes a slight reduction in virulence in the mouse model of yeast cells against a subsequent dose of that same stress (Fig. 2A). systemic candidiasis (Hromatka et al., 2005; Chiranand et al., 2008). For example, acquired thermotolerance has been described in S. Therefore, the nitrosative stress response seems to be most important cerevisiae, S. pombe and more recently in C. albicans (De Virgilio during the early stages of infection when the fungus is battling with et al., 1990; Piper, 1993; Argüelles, 1997). Acquired tolerance has host immune defences. also been observed for oxidative stress in C. albicans (Jamieson et Several common themes are apparent from this brief overview of al., 1996). Acquired stress tolerance is dependent upon the activation key stress responses in C. albicans. First, these stress-signalling of a molecular response to the initial stress, which represents the pathways include regulators that have been highly conserved during induction and accumulation of key proteins or metabolites that fungal evolution. Examples include the Hog1, Mkc1 and Cek1 mediate adaptation to that stress. These proteins and metabolites MAPK modules, and the transcription factors Hsf1 and Cap1. represent a ‘molecular memory’ that can then protect the cell against Second, in comparison with the benign model yeasts S. cerevisiae a subsequent stress, leading to increased survival. However, this and S. pombe, these stress responses have been evolutionarily tuned molecular memory is transient (Leach et al., 2012c), with the length A B Stress Stress Stress 1 Stress 2 Response Response Memory Memory Survival Survival Stress Stress Stress 1 Stress 3 Response Response Memory Memory Survival Survival Time Time Fig. 2. Acquired stress tolerance and stress cross-protection in yeasts. (A) Prior exposure to a stress can protect C. albicans cells against subsequent exposure to that stress (acquired stress tolerance) (upper panel). This indicates the existence of a molecular memory (see ‘Adaptation to sequential stresses’). However, the molecules that represent this memory have biological half-lives. Therefore, this molecular memory is transient, and will be lost during protracted time intervals between stresses (lower panel). (B) In some yeasts, some stresses (stress 1; blue) activate a core transcriptional response (purple) that includes genes that protect against another stress (stress 2; red). In this case, prior exposure to stress 1 often activates a molecular memory that confers protection against stress 2 (upper panel). However, if this core transcriptional response does not include genes that protect against a third stress (stress 3; green), then prior exposure to stress 1 does not activate a relevant molecular memory and does not confer protection against stress 3 (lower panel). The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 of the memory depending upon the decay rates of these proteins and lies much closer to S. cerevisiae than to S. pombe or C. albicans metabolites (Fig. 2A). For example, in the case of thermotolerance, (Roetzer et al., 2008) (Fig. 3). Schizosaccharomyces pombe also the molecular memory in C. albicans probably represents HSPs and displays a core stress response. However, in this case the response is trehalose biosynthetic enzymes rather than the stress protectant driven by Sty1 (Chen et al., 2003), which is the orthologue of the trehalose (Argüelles, 1997; Leach et al., 2012c), because trehalose Hog1 SAPK in S. cerevisiae, C. glabrata and C. albicans (Nikolaou levels decline rapidly once C. albicans cells are returned to lower et al., 2009). In contrast, C. albicans was initially thought to lack a temperatures (Argüelles, 1997). For osmotolerance in C. albicans, core transcriptional response to stress (Enjalbert et al., 2003). the molecular memory is thought to be mediated by glycerol Subsequent work revealed that this yeast does display a core stress biosynthetic enzymes rather than the osmolyte glycerol (You et al., response, but one that comprises a much smaller subset of roughly 25 2012), because glycerol is rapidly extruded from yeast cells when genes (Enjalbert et al., 2006). In C. albicans the roles of Msn2/4-like the osmotic stress is removed (Klipp et al., 2005). By analogy, transcription factors have diverged significantly (Nicholls et al., 2004; acquired tolerance to oxidative stress is probably mediated by the Ramsdale et al., 2008), and the core stress response is coordinated by accumulation of antioxidant enzymes rather than antioxidants Hog1 and Cap1 (Enjalbert et al., 2006). Clearly there has been themselves (Jamieson et al., 1996). significant rewiring of the circuitry that regulates the core stress In some cases, prior exposure to a non-lethal dose of one type of response, as well as of the response itself. stress can also protect yeast cells against a subsequent dose of a This has significant implications for the behaviour of C. albicans different type of stress – a phenomenon called stress cross-protection during exposure to sequential stresses. Thermal stress protects C. (Fig. 2B). For example in S. cerevisiae, a mild heat shock protects albicans against a subsequent oxidative stress, but not against a cells against a subsequent oxidative stress (Wieser et al., 1991; subsequent osmotic or cell wall stress (Enjalbert et al., 2003; Leach Lewis et al., 1995). Similarly, pre-treatment with an oxidative, et al., 2012a). This cross-protection is dependent on Cap1 and osmotic or thermal stress promotes freeze–thaw tolerance in S. correlates with the induction of some Cap1 target genes during heat cerevisiae (Park et al., 1997). The molecular basis for this shock (Nicholls et al., 2009; Leach et al., 2012a). However, this phenomenon lies in the core transcriptional response to stress cross-protection is asymmetric, as an initial treatment with oxidative whereby exposure to any one of several different types of stress stress does not protect C. albicans cells against a subsequent thermal activates genes involved in adaptive responses to many types of stress (Enjalbert et al., 2003; Leach et al., 2012a). stress (Fig. 3). For example, in S. cerevisiae exposure to thermal, These observations are reminiscent of the phenomenon of osmotic, oxidative or pH stress activates several hundred genes with microbial adaptive prediction (Mitchell et al., 2009) (Fig. 4A). roles in stress adaptation, central metabolism and energy generation Mitchell and co-workers argue that some microorganisms inhabit (Gasch et al., 2000; Causton et al., 2001). This core stress response relatively predictable environments, in which one type of is largely dependent on the functionally redundant transcriptional environmental change is often followed by a second type of activators Msn2 and Msn4, which bind to stress response elements stimulus. In such cases organisms may have evolved a regulatory in the promoters of their target genes to mediate their activation circuitry that allows them to predict the second stimulus, thereby (Mager and De Kruijff, 1995; Gasch et al., 2000; Causton et al., conferring an evolutionary advantage. This type of adaptive 2001). Msn2 and its stress-induced transcriptional activation are prediction is displayed by S. cerevisiae, which exploits the elevated downregulated by glucose via the cAMP-protein kinase A (PKA) temperatures associated with vigorous fermentation to induce signalling pathway (Görner et al., 1998; Garreau et al., 2000). oxidative stress genes that will be required once glucose is An analogous Msn2-dependent core transcriptional response to exhausted and cells switch to respiratory and oxidative metabolism stress is displayed by Candida glabrata, which in evolutionary terms (Mitchell et al., 2009). This adaptive prediction is asymmetric, as >400 MYA ~150 MYA ~60 MYA S. cerevisiae C. glabrata C. albicans S. pombe Oxidative Osmotic Oxidative Osmotic Oxidative Osmotic Oxidative Osmotic Glucose Heavy Heat pH Heat Heat starvation Heavy metal metal Msn2/4 Msn2/4 Sty1 CSR CSR CSR CSR ~220 genes ~400 genes ~20 genes ~140 genes Fig. 3. Core stress responses in yeasts. The yeasts C. albicans, Saccharomyces cerevisiae, Candida glabrata and Schizosaccharomyces pombe are evolutionarily separated by many millions of years and occupy contrasting niches: green, environmental niches; red, pathogens. Three of these yeasts display core transcriptional responses to stress in which relatively large numbers of genes are commonly induced in response to different stresses. In S. cerevisiae and C. glabrata the zinc finger transcription factors Msn2 and Msn4 contribute significantly to the core stress response, whereas this response in S. pombe is driven by the Sty1 SAPK. The core transcriptional response has diverged significantly in C. albicans, in which there is a relatively small number of core stress genes (see ‘Adaptation to sequential stresses’). The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Stress 1 Stress 2 Stress 1 Stress 2 Response 1 Response 2 Response 1 Response 2 Asymmetric Symmetric adaptive prediction adaptive prediction Elevated Oxidative Elevated Oxidative Glucose temperature stress temperature stress exposure HSP Oxidative HSP Oxidative Metabolic genes stress genes genes stress genes genes S. cerevisiae C. albicans Fig. 4. Anticipatory prediction in C. albicans and S. cerevisiae. (A) As described by Mitchell and co-workers, microbes often display adaptive prediction, whereby exposure to one environmental input can lead to the anticipatory induction of the response to a second environmental input (Mitchell et al., 2009). The authors argue that this provides an evolutionary advantage to the microbe because the first input is often followed by the second input in its normal environmental niche. Anticipatory responses can be asymmetric or symmetric. (B) Saccharomyces cerevisiae displays asymmetric anticipatory adaptive prediction by activating oxidative stress genes in response to elevated temperatures. Candida albicans displays an analogous asymmetric anticipatory adaptive response (Mitchell et al., 2009). This pathogen also displays symmetric anticipatory adaptive prediction by activating oxidative stress genes in response to glucose exposure and by activating carbohydrate metabolism in response to oxidative stress (see ‘Adaptation to sequential stresses’). oxidative stress does not induce heat shock gene expression in S. Adaptation to combinatorial stresses cerevisiae (Mitchell et al., 2009). An analogous asymmetric As mentioned above, C. albicans cells are often simultaneously relationship between oxidative and heat shock gene regulation is exposed to multiple stresses within the complex host niches they observed in C. albicans: in general, oxidative stress functions are inhabit. Possibly the best example of combinatorial stress occurs induced in response to heat shock, but heat shock genes are not following phagocytosis by neutrophils or macrophages, when the induced by an oxidative stress (Enjalbert et al., 2003) (Fig. 4B). This fungus is bombarded with ROS, RNS and cationic fluxes is consistent with the idea that adaptive prediction might have (Rementería et al., 1995; Vázquez-Torres and Balish, 1997; Brown, evolved in C. albicans such that the pathogen anticipates oxidative 2011; Nüsse, 2011). However, combinatorial stresses are likely to attack by phagocytic cells in response to fevers associated with be relevant in many other host niches, such as during mucosal inflammatory responses. invasion (where oxidative stresses are encountered while adjusting A second example of adaptive prediction has been described in C. cellular water balance) and kidney infection (where respiring cells albicans. In this fungus, oxidative stress genes are activated must deal with endogenous ROS while adapting to relatively high following exposure to glucose, thereby conferring elevated salt concentrations). How do C. albicans cells respond to such resistance to acute oxidative stress (Rodaki et al., 2009) (Fig. 4B). combinatorial stresses? We have predicted that the adaptive This phenomenon does not depend on Hog1 or Cap1 (Rodaki et al., responses to such combinatorial stresses might not be equivalent to 2009). Instead, glucose-enhanced oxidative stress resistance appears the sum of the responses to the corresponding individual stresses to be regulated by evolutionarily conserved glucose signalling (Kaloriti et al., 2012). Our rationale is that unexpected cross-talk pathways (I.B. and A.J.P.B., unpublished). This anticipatory between the relevant signalling pathways might exist. Several response, which is triggered by the glucose concentrations present examples of this have emerged recently. in the bloodstream, is likely to be relevant in the disease context. Combinatorial oxidative (H O ) plus nitrosative stresses 2 2 Candida albicans cells that enter the bloodstream are exposed to (dipropylenetriamine-NONOate, DPTA-NONOate) and glucose, and this may help to protect them against the impending combinatorial cationic (NaCl) plus nitrosative stresses appear to attack from phagocytic cells. If this were true, the phenomenon of exert additive effects upon the growth of C. albicans cells (Kaloriti glucose-enhanced oxidative stress resistance must have evolved et al., 2012). However, YHB1 gene induction is attenuated under relatively recently. This appears to be the case (I.B. and A.J.P.B., these conditions, indicating that Cta4 signalling is compromised unpublished). Indeed, the opposite phenotype is observed in S. (A.T. and A.J.P.B., unpublished). Significantly, non-additive effects cerevisiae: glucose reduces stress resistance in this benign yeast, are observed for combinatorial cationic plus oxidative stresses which has evolved in environmental niches (Mager and De Kruijff, (Kaloriti et al., 2012). These stresses kill C. albicans synergistically. 1995; Görner et al., 1998; Garreau et al., 2000). The basis for this appears to be ‘stress pathway interference’, This anticipatory response appears to be symmetric because whereby both Cap1 and Hog1 signalling are compromised by the exposing C. albicans cells to hydrogen peroxide leads to the combination of cationic and oxidative stress. As a result, cationic activation of genes involved in central carbon metabolism (Enjalbert and oxidative stress genes are not induced, and intracellular ROS et al., 2006). However, this particular response (oxidative stress- levels increase, leading to cell death (D.K., M.D.J., A.T. and induced metabolic activation), which is conserved in other yeasts A.J.P.B., unpublished). Indeed, hydrogen peroxide has been shown (Gasch et al., 2000; Causton et al., 2001; Chen et al., 2003; Enjalbert to stimulate apoptotic cell death in C. albicans via Ras-cAMP et al., 2006), may have less to do with anticipatory prediction and signalling (Phillips et al., 2003; Phillips et al., 2006). This appears more to do with the need for metabolic intermediates and energy to to be highly relevant to host–fungus interactions because the drive oxidative stress adaptation (Brown et al., 2012a). effective killing of C. albicans cells by human neutrophils appears The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 to depend on the extreme potency of combinatorial cationic plus (reviewed by Brown et al., 2009). We have now shown that oxidative stresses (D.K., M.D.J., A.T. and A.J.P.B., unpublished). combinatorial effects can also be triggered at the biochemical level. Combinatorial effects are also observed between thermal and In this case the inhibition of key detoxification functions by cationic other stresses. For example, elevated temperatures decrease the stresses leads to the build up of intracellular ROS, causing stress sensitivity of C. albicans cells to a cell wall stress (Calcofluor pathway interference and ultimately cell death (D.K., M.D.J., A.T. White), but have little effect upon osmo-sensitivity (Leach et al., and A.J.P.B., unpublished). In addition, we have shown that 2012a). This stress interaction appears to be mediated via Hsp90 combinatorial effects can be mediated by a biological transistor. In (Leach et al., 2012a). As described above, the Hog1, Mkc1 and this case, Hsp90 coordinates the activities of multiple signalling Cek1 pathways modulate cell wall functionality. The MAP kinases pathways involved in cellular adaptation (Leach et al., 2012b). in these pathways are all client proteins of Hsp90, and their While the responses of fungal cells to individual stresses are now activation is modulated by Hsp90 (Leach et al., 2012a). Temperature reasonably well understood, little is known about the mechanisms fluctuations have been shown to influence HSP90 expression levels that underlie combinatorial stress adaptation. Yet, combinatorial as well as the binding of Hsp90 to its client proteins in C. albicans stress adaptation is highly relevant to natural environments. (Nicholls et al., 2009; Diezmann et al., 2012; Leach et al., 2012a; Leach et al., 2012c). Furthermore, ambient temperature affects the Impact of dynamic host niches upon stress adaptation cell wall proteome, and Hsp90 depletion alters cell wall architecture Metabolic changes within host niches also affect stress adaptation in (Leach et al., 2012a; Heilmann et al., 2013). Therefore, Hsp90 has C. albicans (Fig. 6). In particular, many host niches either lack been proposed to act as a biological transistor that tunes sugars such as glucose or contain glucose at low concentrations. environmental responses, including cell wall remodelling, to the Instead, these niches contain complex mixtures of alternative carbon ambient temperature of the cell (Leach et al., 2012b). sources such as amino acids, carboxylic acids such as lactate, and Therefore, as predicted (Kaloriti et al., 2012), combinatorial fatty acids. Consequently, C. albicans must assimilate these stresses exert unexpected effects upon the classical regulatory alternative carbon sources if it is to grow and colonise these niches. pathways that mediate responses to specific stresses (Fig. 1). The Not surprisingly, metabolic pathways that are essential for the available data have revealed several distinct molecular mechanisms assimilation of these alternative carbon sources, such as by which combinatorial cross-talk can occur (Fig. 5). First, there gluconeogenesis and the glyoxylate cycle, are required for full appears to be signalling cross-talk between the MAPKs in critical virulence (Lorenz and Fink, 2001; Barelle et al., 2006; Piekarska et stress signalling pathways. This is suggested by mutational analyses al., 2006; Ramírez and Lorenz, 2007). Furthermore, lactate whereby the deletion of HOG1 leads to the derepression of Cek1 assimilation is essential for C. glabrata to colonise the intestine phosphorylation and the inhibition of Mkc1 phosphorylation (Arana (Ueno et al., 2011), and a significant proportion of C. albicans cells et al., 2005). Cross-talk also exists at the chemical level. infecting the kidney activate pathways for alternative carbon Combinations of H O and NaCl lead to the formation of utilisation (Barelle et al., 2006), as do phagocytosed C. albicans 2 2 hypochlorous acid (HOCl), and nitric oxide and superoxide react to cells (Lorenz et al., 2004; Fradin et al., 2005; Barelle et al., 2006; form peroxynitrite (ONOO ), and nitrite and hypochlorous acid Miramón et al., 2012). The metabolic activity of C. albicans can combine to form nitryl chloride (NO Cl), generating cocktails of modify the pH of its microenvironment (Vylkova et al., 2011) toxic compounds that can damage lipids, proteins and nucleic acids adding to the dynamism of host niches. The regulatory circuitry that Temperature A B Bck1 Ste11 Ssk2 Hsp90 Hst7 Pbs2 Mkk1 Hsf1 Cek1 Hog1 Mck1 Hog1 Cek1 Mck1 Thermotolerance Cationic Oxidative C D plus stress stress superoxide H O 2 2 NO ONOO hydrogen nitric peroxide peroxynitrite HOCl oxide Cationic Oxidative hypochlorous stress genes stress genes acid RCS ROS RNS Stress genes Fig. 5. Mechanisms underlying combinatorial stress effects in C. albicans. Several distinct mechanisms contribute to combinatorial stress effects in C. albicans (see ‘Adaptation to combinatorial stresses’). (A) Classical cross-talk occurs between the MAPK signalling pathways (Alonso Monge et al., 2006). Hog1 signalling pathway: Ssk2, MAPKKK; Pbs2, MAPKK; Hog1, MAPK/SAPK. Cell integrity pathway: Bck1, MAPKKK; Mkk1, MAPKK; Mkc1, MAPK. Mating/invasive growth pathway: Ste11, MAPKKK; Hst7, MAPKK; Cek1, MAPK. (B) Hsp90 acts as a biological transistor, modulating the activities of the transcription factor Hsf1 and the MAPKs in response to thermal fluctuations (Leach et al., 2012a; Leach et al., 2012b). (C) Combinatorial cationic plus oxidative stress leads to stress pathway interference, whereby Hog1 and Cap1 signalling are affected by oxidative and cationic stress, respectively (D.K., M.D.J., A.T. and A.J.P.B., unpublished). (D) There is cross-talk at the chemical level, whereby different reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive chlorine species (RCS) can be generated spontaneously and by enzymatic catalysis (Brown et al., 2009; Brown et al., 2011), presumably leading to the activation of different subsets of stress genes. The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Carbon source Proteome Architecture Cell wall Biophysical properties Glucose Lactate Stress Immune adaptation recognition Virulence Fig. 6. Impact of carbon source on C. albicans. Changes in carbon source affect the proteome, architecture and biophysical properties of the C. albicans cell wall. This affects stress adaptation, immune recognition and virulence (Ene et al., 2012a; Ene et al., 2012b; Ene et al., 2013). Transmission electron micrographs of cell walls from C. albicans cells grown on glucose or lactate are shown on the right. regulates carbon assimilation in C. albicans has undergone individual cells vary even within specific host niches. Therefore, the evolutionary rewiring (Ihmels et al., 2005; Martchenko et al., 2007; spatial regulation of stress adaptation must also be examined during Lavoie et al., 2009; Sandai et al., 2012), just as is the case for stress infection. This must either be done by examining the responses of adaptation (discussed above). individual cells in vivo, for example using GFP-based single-cell Despite the fact that glucose is limiting or absent in many host profiling methods (Barelle et al., 2006; Enjalbert et al., 2007; niches, most studies of stress adaptation in C. albicans have been Miramón et al., 2012), or by increasing the sensitivity of RNA performed on cells grown in media containing 2% glucose. sequencing technologies and increasing their spatial resolution, for Recently, we showed that growth on physiologically relevant example by exploiting laser capture microscopy. These approaches alternative carbon sources, such as lactate or oleic acid, affects stress are being pursued by the Aberdeen Fungal Group (J.P., S.S. and adaptation in C. albicans (Ene et al., 2012a). Lactate-grown cells are A.J.P.B., unpublished). more resistant to osmotic stress, cell wall stresses and some In addition, at least three aspects of stress adaptation that are of antifungal drugs. This increased stress resistance is not dependent direct relevance in vivo need further dissection in vitro. First, which on Hog1 or Mkc1 signalling. Instead, it relates to the effects of anticipatory responses in C. albicans influence host colonisation and alternative carbon sources on the proteomic content and architecture disease progression, and how are these anticipatory responses of the cell wall, which in turn impact upon the biophysical properties controlled at the molecular level? Second, which combinatorial of the cell wall (Ene et al., 2012a; Ene et al., 2012b) (Fig. 6). These stress responses in C. albicans influence host–fungus interactions, alterations at the cell surface affect host recognition of C. albicans and how are they regulated? Third, how does metabolic adaptation cells and influence the virulence of this pathogen in both systemic influence stress resistance within host niches? Despite the limited and mucosal models of infection (Ene et al., 2012a; Ene et al., exploration of these issues, it is already clear that they involve non- 2013). Clearly, metabolic adaptation affects stress responses in C. additive behaviours that reflect unexpected signalling, albicans, and this further complicates our understanding of transcriptional, biochemical and chemical cross-talk. Furthermore environmental adaptation of this fungus within the complex and many of these responses are dynamic and dose dependent. Given dynamic microenvironments it occupies during host colonisation their complexity, a combination of experimental approaches and and disease progression. Significantly, this is also likely to affect the predictive mathematical modelling seems especially important for efficacy of antifungal drug treatments against individual C. albicans the development of a true understanding of these adaptive processes. cells in these niches (Ene et al., 2012a). Such studies will provide important insights into the forces that have driven the recent evolution of this pathogen in its host. Outlook In closing, it is worth emphasising that studies of stress adaptation Significant advances have been made in our understanding of stress are revealing points of fragility in C. albicans that could potentially adaptation in C. albicans, and progress is being made towards the provide targets for translational research directed towards the elaboration of specific stress signalling pathways. This is important development of novel antifungal therapies. Indeed, the therapeutic because stress adaptation contributes to the virulence of this major potential of Hsp90 inhibitors is being pursued by a number of fungal pathogen of humans. However, host niches are complex and laboratories (Dolgin and Motluk, 2011). Therefore, observations dynamic, and the impact of this complexity and dynamism upon such as the acute sensitivity of C. albicans towards combinatorial stress adaptation remains largely unexplored. In particular, how are cationic plus oxidative stress could, in principle, be exploited stress responses regulated temporally during host colonisation and therapeutically. disease progression? The elegant microarray studies performed by Bernie Hube’s group go some of the way to addressing this question Acknowledgements We thank our friends and colleagues in the Aberdeen Fungal Group, the CRISP (Fradin et al., 2005; Thewes et al., 2007; Zakikhany et al., 2007; Consortium, the FINSysB Network and the Cowen laboratory for stimulating Wilson et al., 2009). However, microarray studies average the discussions and helpful advice. Neil Gow, Frank Odds, Carol Munro, Gordon molecular behaviour of the fungal population as a whole, and fungal Brown, Janet Quinn, Ken Haynes, Christophe d’Enfert, Bernard Hube, Mihai populations display heterogeneous behaviours in host niches Netea, Frans Klis, Leah Cowen, Stephanie Diezmann and Joe Heitman deserve (Barelle et al., 2006). This is because the microenvironments of special mention. The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Competing interests of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459, 657-662. The authors declare no competing financial interests. Calderone, R. (2002). Candida and Candidiasis. Washington, DC: ASM Press. Calderone, R. A. and Clancy, C. J. (2012). Candida and Candidiasis, 2nd edn. Author contributions Washington, DC: ASM Press. All authors contributed to the writing of this review, the initial draft being prepared Cantero, P. D. and Ernst, J. F. (2011). Damage to the glycoshield activates PMT- by A.J.P.B. and M.D.L. directed O-mannosylation via the Msb2-Cek1 pathway in Candida albicans. Mol. Microbiol. 80, 715-725. Funding Causton, H. C., Ren, B., Koh, S. S., Harbison, C. T., Kanin, E., Jennings, E. G., Lee, T. I., True, H. L., Lander, E. S. and Young, R. A. (2001). Remodeling of yeast We are grateful to our funding bodies for their support. This work was supported by genome expression in response to environmental changes. Mol. Biol. Cell 12, 323- the European Commission [FINSysB, PITN-GA-2008-214004; STRIFE, ERC- 2009-AdG-249793], by the UK Biotechnology and Biological Research Council Chauhan, N., Inglis, D., Roman, E., Pla, J., Li, D., Calera, J. A. and Calderone, R. [grant numbers BBS/B/06679; BB/C510391/1; BB/D009308/1; BB/F000111/1; (2003). Candida albicans response regulator gene SSK1 regulates a subset of BB/F010826/1; BB/F00513X/1], and by the Wellcome Trust [grant numbers genes whose functions are associated with cell wall biosynthesis and adaptation to 080088, 097377]. M.D.L. was also supported by a Carnegie/Caledonian oxidative stress. Eukaryot. Cell 2, 1018-1024. Scholarship and a Sir Henry Wellcome Postdoctoral Fellowship from the Wellcome Chauhan, N., Latge, J. P. and Calderone, R. A. (2006). Signalling and oxidant Trust [grant number 096072]. 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T., Boucher, G., Rogers, P. Engineered control of cell morphology in vivo reveals distinct roles for yeast and D. and Raymond, M. (2009). Identification of the Candida albicans Cap1p regulon. filamentous forms of Candida albicans during infection. Eukaryot. Cell 2, 1053-1060. Eukaryot. Cell 8, 806-820. The Journal of Experimental Biology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Biology The Company of Biologists

Stress adaptation in a pathogenic fungus

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© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 144-155 doi:10.1242/jeb.088930 REVIEW Alistair J. P. Brown*, Susan Budge, Despoina Kaloriti, Anna Tillmann, Mette D. Jacobsen, Zhikang Yin, ‡ § ¶ Iuliana V. Ene , Iryna Bohovych , Doblin Sandai , Stavroula Kastora, Joanna Potrykus, Elizabeth R. Ballou, Delma S. Childers, Shahida Shahana and Michelle D. Leach** ABSTRACT normally exists as a harmless commensal organism in the microflora of the skin, oral cavity, and gastrointestinal and urogenital tracts of Candida albicans is a major fungal pathogen of humans. This yeast most healthy individuals (Odds, 1988; Calderone, 2002; Calderone is carried by many individuals as a harmless commensal, but when and Clancy, 2012). However, C. albicans frequently causes oral and immune defences are perturbed it causes mucosal infections vaginal infections (thrush) when the microflora is disturbed by (thrush). Additionally, when the immune system becomes severely antibiotic usage or when immune defences are perturbed, for compromised, C. albicans often causes life-threatening systemic example in HIV patients (Sobel, 2007; Revankar and Sobel, 2012). infections. A battery of virulence factors and fitness attributes promote In individuals whose immune systems are severely compromised the pathogenicity of C. albicans. Fitness attributes include robust (such as neutropenic patients undergoing chemotherapy or transplant responses to local environmental stresses, the inactivation of which surgery), the fungus can survive in the bloodstream, leading to the attenuates virulence. Stress signalling pathways in C. albicans colonisation of internal organs such as the kidney, liver, spleen and include evolutionarily conserved modules. However, there has been brain (Pfaller and Diekema, 2007; Calderone and Clancy, 2012). rewiring of some stress regulatory circuitry such that the roles of a Candida is the fourth most common cause of hospital-acquired number of regulators in C. albicans have diverged relative to the bloodstream infections, over half of which can be fatal in some benign model yeasts Saccharomyces cerevisiae and patient groups (Perlroth et al., 2007). This high morbidity exists Schizosaccharomyces pombe. This reflects the specific evolution of despite the availability of specialised antifungal drugs such as the C. albicans as an opportunistic pathogen obligately associated with azoles, polyenes and echinocandins (Odds et al., 2003a; Brown et warm-blooded animals, compared with other yeasts that are found al., 2012b), reflecting the challenges in diagnosing systemic fungal across diverse environmental niches. Our understanding of C. infections, the resultant delays in treatment, and the limited choice albicans stress signalling is based primarily on the in vitro responses of effective antifungal drugs (Pfaller and Diekema, 2010; Brown et of glucose-grown cells to individual stresses. However, in vivo this al., 2012b). From the fungal perspective, it is clear that C. albicans pathogen occupies complex and dynamic host niches characterised can adapt effectively to diverse host niches. by alternative carbon sources and simultaneous exposure to The evolutionary history of C. albicans has established both its combinations of stresses (rather than individual stresses). It has pathogenic behaviour and also its properties as an experimental become apparent that changes in carbon source strongly influence system. Candida albicans is a member of the ascomycete phylum, stress resistance, and that some combinatorial stresses exert non- which includes the model yeasts Saccharomyces cerevisiae and additive effects upon C. albicans. These effects, which are relevant Schizosaccharomyces pombe. These benign model yeasts provide to fungus–host interactions during disease progression, are mediated paradigms against which C. albicans is often compared (Berman by multiple mechanisms that include signalling and chemical and Sudbery, 2002; Enjalbert et al., 2006; Noble and Johnson, crosstalk, stress pathway interference and a biological transistor. 2007). However, in evolutionary terms C. albicans is only distantly KEY WORDS: Candida albicans, Fungal pathogenicity, Heat shock, related to S. cerevisiae (circa 150 million years) and S. pombe (>400 Oxidative stress, Nitrosative stress, Osmotic stress, Cationic million years) (Galagan et al., 2005), and the latter evolutionary stress, Stress adaptation, Carbon metabolism distance represents greater separation than exists between humans and sharks. Furthermore, although ascomycetes are generally Introduction: Candida albicans – an opportunistic pathogen defined by their packaging of sexual spores into an ascus structure, of humans C. albicans has not been observed to undergo meiosis to generate Candida albicans is a major fungal pathogen of humans that spores. Rather, this diploid yeast, which until very recently was occupies a wide range of divergent niches within the host. It thought to be constitutively diploid (Hickman et al., 2013), displays a complex parasexual cycle. Candida albicans must undergo homozygosis at the mating type locus (MTL) and then undergo an School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. epigenetic switch to mating competent cells (the opaque form) Present address: Department of Molecular Microbiology and Immunology, Brown before it mates to form tetraploids (Noble and Johnson, 2007). This University, Providence, RI 02912, USA. Present address: Nebraska Redox is followed by chromosome loss to return to the diploid state Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588-0662, USA. Present address: Institut Perubatan and Pergigian Termaju, Universiti Sains (Forche et al., 2008). While parasexual recombination could have Malaysia, Pulau Pinang, Malaysia. **Present address: Department of Molecular contributed to the recent evolution of C. albicans, the population Genetics, University of Toronto, Medical Sciences Building, Toronto, Canada, structure is predominantly clonal (Cowen et al., 2002; Odds et al., M5S 1A8. 2007). Indeed, its recent evolution appears to have been driven *Author for correspondence ([email protected]) largely by its clonal behaviour as a pathogen. Candida albicans has not been associated with any particular environmental niche and This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted hence is thought to be obligately associated with warm-blooded use, distribution and reproduction in any medium provided that the original work is properly attributed. animals (Odds, 1988). Therefore, it is not surprising that this fungus The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 the host. For example, oxidative, nitrosative and heat shock List of abbreviations functions are induced when cells are phagocytosed by macrophages GFP green fluorescent protein or neutrophils (Rubin-Bejerano et al., 2003; Lorenz et al., 2004; HSE heat shock element Fradin et al., 2005), and the niche-specific induction of specific HSP heat shock protein stress responses has been confirmed by single cell profiling using MAPK mitogen-activated protein kinase diagnostic green fluorescent protein (GFP) fusions (Enjalbert et al., MAPKK MAP kinase kinase MAPKKK MAP kinase kinase kinase 2007; Miramón et al., 2012). Second, the virulence of C. albicans RCS reactive chlorine species in mouse models of infection is attenuated by the inactivation of key RNS reactive nitrogen species stress functions such as the stress-activated protein kinase (SAPK) ROS reactive oxygen species Hog1, the catalase Cat1 or the superoxide dismutase Sod1 (Wysong SAPK stress-activated protein kinase et al., 1998; Alonso-Monge et al., 1999; Hwang et al., 2002; Cheetham et al., 2011). Significant progress has been made in the has undergone the rapid evolution of virulence factors and fitness elaboration of stress-adaptive responses, their regulation in C. attributes associated with its pathogenicity (Butler et al., 2009; albicans and their divergence from the corresponding pathways in Nikolaou et al., 2009) as well as evolutionary rewiring of model yeasts. A brief overview of these mechanisms will be transcriptional and post-transcriptional circuitries relative to S. discussed here. This provides the platform for the main theme of this cerevisiae (Ihmels et al., 2005; Martchenko et al., 2007; Lavoie et review – stress adaptation in the context of complex and dynamic al., 2009; Baker et al., 2012; Sandai et al., 2012). These changes host niches (mentioned above), in which C. albicans cells must have had a significant impact on the evolution of stress adaptation respond to multiple environmental inputs, rather than to the in C. albicans (Brown et al., 2012a). individual stresses commonly studied in vitro. The loss of a bona fide sexual cycle has had a major impact on the experimental dissection of C. albicans pathobiology. Researchers Overview of stress adaptation mechanisms in C. albicans have had to rely mainly on genomic and molecular approaches, Stress signalling pathways are relatively well characterised in S. rather than genetic strategies to examine the virulence of this fungus cerevisiae and S. pombe. A number of the key regulators are (Noble and Johnson, 2007). Nevertheless, these approaches have evolutionarily conserved in C. albicans (Butler et al., 2009; revealed an armoury of virulence factors that promote the Nikolaou et al., 2009) (Fig. 1). However, the roles of some of these pathogenicity of C. albicans. Virulence factors have been defined as regulators have diverged (Enjalbert et al., 2003; Nicholls et al., those fungal factors that interact directly with host components 2004; Ramsdale et al., 2008; Cheetham et al., 2007), and C. albicans (Odds et al., 2003b). For example, reversible morphogenetic is relatively resistant to physiologically relevant stresses compared transitions between yeast, pseudohyphal and hyphal growth forms with model yeasts (Jamieson et al., 1996; Nikolaou et al., 2009). contribute to the virulence of C. albicans (Lo et al., 1997; Saville et This is consistent with the idea that stress responses in C. albicans al., 2003). Yeast forms are thought to promote dissemination, have been evolutionarily tuned to host niches. Stress signalling in C. whereas the filamentous forms are better suited to penetrate tissue. albicans has been described in a number of recent reviews (Chauhan Hyphae also display thigmotropic responses that appear to et al., 2006; Alonso-Monge et al., 2009b; Brown et al., 2009; Smith contribute to tissue penetration (Sherwood et al., 1992; Brand, et al., 2010; Brown et al., 2012a). Therefore, the purpose of this 2012). Initial colonisation is mediated by families of cell surface section is to summarise key stress signalling pathways, highlighting adhesins that promote adherence to host tissues (Staab et al., 1999; their relevance to infection. Hoyer et al., 2008). One of these adhesins, Als3, also acts as an invasin by promoting the invasion of endothelial cells (Phan et al., Heat shock 2007), contributing to the assimilation of the essential micronutrient The heat shock response is ubiquitous in nature. In eukaryotes, it iron (Almeida et al., 2008; Almeida et al., 2009) and to brain involves the induction of a defined set of heat shock proteins colonisation (Liu et al., 2011). Candida albicans expresses (HSPs), many of which promote the folding of client proteins or additional factors involved in iron and zinc assimilation, some of target aggregated or damaged proteins for degradation (Parsell and which are essential for virulence (Almeida et al., 2009; Citiulo et al., Lindquist, 1993; Feder and Hofmann, 1999). The response in C. 2012), and which are induced during renal infection (J.P. and albicans, as in other yeasts, is driven by the heat shock transcription A.J.P.B., unpublished). Candida albicans also secretes families of factor Hsf1 (Nicholls et al., 2009). Hsf1 is conserved from yeasts to hydrolytic enzymes including proteases, lipases and phospholipases humans and is essential for viability (Sorger and Pelham, 1988; (Naglik et al., 2003; Schaller et al., 2005) that enhance tissue Sarge et al., 1993; Wu, 1995). In response to acute heat shock, C. invasion, provide nutrients to support fungal growth and modulate albicans Hsf1 becomes phosphorylated and induces the expression host immune responses (Pietrella et al., 2010). These and other of target heat shock protein (HSP) genes via canonical heat shock factors are temporally and spatially regulated during colonisation elements (HSEs) in their promoters (Nicholls et al., 2009), an and disease progression, thereby enhancing C. albicans interaction that is conserved in other eukaryotes (Sorger and Pelham, pathogenicity. 1988; Jakobsen and Pelham, 1988; Holmberg et al., 2001). HSP Additional factors promote the virulence of C. albicans without gene induction leads to the refolding or degradation of damaged interacting directly with the host. These factors, which have been proteins, thereby promoting cellular adaptation to the thermal insult. termed ‘fitness attributes’ (Brown, 2005), include functions involved Indeed, in C. albicans heat shock induces polyubiquitin (UBI4) in metabolic and stress adaptation and act by tuning the expression, which is required for resistance to thermal stress (Roig physiological fitness of C. albicans cells to their local host and Gozalbo, 2003; Leach et al., 2011). The HSP90 gene is also microenvironment. Two main types of evidence have highlighted the activated in an Hsf1-dependent fashion (Nicholls et al., 2009). Heat importance of stress adaptation for the virulence of C. albicans. shock protein 90 (Hsp90) has been described as a molecular First, numerous genome-wide expression profiles have demonstrated transistor as it modulates the activity of client regulatory proteins that stress genes are induced when the fungus comes in contact with (Leach et al., 2012a). Following thermal adaptation, Hsp90 interacts The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Osmotic/ Enjalbert et al., 2006). This leads to the accumulation of glycerol, Thermal Nitrosative Oxidative cationic Cell wall damage stress stress stress the restoration of turgor pressure and the resumption of growth. stress Glycerol biosynthetic gene induction, glycerol accumulation and the successful adaptation of C. albicans cells to osmotic/cationic Ssk2 Bck1 Ste11 stresses are Hog1 dependent (San José et al., 1996; Smith et al., Pbs2 Mkk1 Hst7 2004). Hog1 Mck1 Cek1 Hog1 is a component of a highly conserved mitogen-activated Hsp90 protein (MAP) kinase pathway involved in osmo-adaptation in other Hsf1 Cta4 Cap1 Skn7 yeasts (Nikolaou et al., 2009; Smith et al., 2010). In C. albicans, this MAP kinase (MAPK) is activated by the MAP kinase kinase Glutaredoxin SODs & thioredoxin (MAPKK) Pbs2, which in turn is activated by a single MAP kinase catalase Glycerol Chaperones Yhb1 Cell wall remodelling systems accumulation kinase kinase (MAPKKK), Ssk2 (Arana et al., 2005; Cheetham et Glutathione Trehalose al., 2007) (Fig. 1). However, the upstream regulators that activate this MAPK module in response to osmotic stress have not been Stress adaptation established unambiguously in C. albicans. In S. cerevisiae, this Virulence MAPK module responds to two well-defined upstream branches (reviewed by Smith et al., 2010). The Sho1 branch activates Hog1 Fig. 1. Conserved stress regulators in Candida albicans. Evolutionarily signalling via Cdc42, Ste50, Ste20 and Cla4, and through the conserved mitogen-activated protein kinase (MAPK) signalling molecules MAPKKK Ste11 specifically in response to heat or osmotic stress. (red) and transcription factors (blue) contribute to the regulation of stress The Sln1 phospho-relay system includes Ypd1 and Ssk1, and functions in C. albicans (see ‘Overview of stress adaptation mechanisms in activates Hog1 signalling via the MAPKKKs Ssk2 and Ssk22 in C. albicans’). Hsf1 and Hsp90 operate in an autoregulatory circuit, whereby response to a broad range of stresses, including osmotic stress. synthesis of the biological transistor Hsp90 (green) is activated by Hsf1 in Candida albicans has orthologues for many of these proteins response to heat shock, and Hsp90 then downregulates Hsf1 (see ‘Overview of stress adaptation mechanisms in C. albicans’). These pathways are (Nikolaou et al., 2009), as well as proteins that are related to represented as linear pathways (for simplicity), but most probably operate in histidine kinases in S. cerevisiae and S. pombe (C. albicans Sln1, an integrated network. Heat shock pathway: Hsp90, heat shock protein 90; Chk1, Nik1) (Kruppa and Calderone, 2006). However, in C. Hsf1, heat shock transcription factor. Nitrosative stress pathway: Cta4, zinc albicans none of these histidine kinases or Ssk1 is essential for the cluster transcription factor; Yhb1, nitric oxide dioxygenase. Oxidative stress osmotic stress-induced activation of Hog1 (Chauhan et al., 2003; pathway: Cap1, AP-1 bZIP transcription factor; Skn7; putative response Kruppa and Calderone, 2006), suggesting that the Sln1 branch does regulator; SODs, superoxide dismutases. Hog1 signalling pathway: Ssk2, not transduce osmotic stress signals to Hog1. Furthermore, a ypd1 MAPK kinase kinase (MAPKKK); Pbs2, MAPK kinase (MAPKK); Hog1, MAPK/stress-activated protein kinase (SAPK). Cell integrity pathway: Bck1, sho1 double mutation does not block osmotic stress signalling to MAPKKK; Mkk1, MAPKK; Mkc1, MAPK. Mating/invasive growth pathway: Hog1 in C. albicans (Román et al., 2005), indicating that the Sho1 Ste11, MAPKKK; Hst7, MAPKK; Cek1, MAPK. branch is not essential for osmotic stress signalling either. Therefore, it is not yet clear how osmotic stress signals are transduced to Hog1, and there appears to have been significant evolutionary rewiring of physically with Hsf1 to downregulate the heat shock response in C. the upstream regulators of this stress pathway. albicans (Leach et al., 2012b) (Fig. 1). The inactivation of Hog1 attenuates the virulence of C. albicans Significantly, while other conserved stress regulatory circuits have (Alonso-Monge et al., 1999; Cheetham et al., 2011). However, this undergone evolutionary rewiring (see below), heat shock regulation is not attributable simply to the loss of osmotic or cationic stress has been maintained in C. albicans (Nicholls et al., 2009) despite its adaptation because Hog1 has been shown to execute additional obligate association with warm-blooded animals (Odds, 1988). functions. Hog1 is required for adaptation to other stresses, Presumably the fungus occupies thermally buffered niches in the modulates cellular morphogenesis, influences metabolism and host and is generally sheltered from the acute heat shocks that are affects cell wall functionality (Alonso-Monge et al., 1999; Alonso- imposed in the laboratory. Interestingly, mutations that block the Monge et al., 2003; Alonso-Monge et al., 2009a; Smith et al., 2004; activation of the heat shock response attenuate the virulence of C. Eisman et al., 2006). Nevertheless, several observations suggest that albicans (Nicholls et al., 2011). Mathematical modelling of the osmotic and cationic stress adaptation play significant roles in dynamic regulation of Hsf1 during thermal adaptation has provided certain host niches. First, NaCl concentrations can approach −1 an answer to this conundrum (Leach et al., 2012c). The Hsf1–HSE 600 mmol l in the kidney and be high in the urine (Ohno et al., regulon appears to be activated even during slow thermal transitions 1997; Zhang et al., 2004). Second, C. albicans cells are exposed to such as those suffered by febrile patients. This explains why Hsf1 K fluxes following phagocytosis by host immune cells (Da Silva- activation is essential for the virulence of C. albicans (Nicholls et Santos et al., 2002; Fang, 2004). Third, mathematical modelling of al., 2011). Clearly, the Hsf1–HSE regulon is critical for the osmotic stress adaptation in S. cerevisiae has highlighted the role of maintenance of thermal homeostasis, not merely for adaptation to this regulatory apparatus in mediating cellular osmo-homeostasis acute heat shocks. and the maintenance of water balance (Klipp et al., 2005), in addition to its role in adaptation to the acute osmotic shocks that Osmotic and cationic stress experimentalists tend to impose in vitro. Hence, Hog1-mediated Exposure to NaCl or KCl imposes osmotic and cationic stress, osmotic adaptation is likely to be required in many host niches. which causes rapid water loss, a reduction in cell size and loss of turgor pressure (Kühn and Klipp, 2012). This triggers the Cell wall stress phosphorylation and nuclear accumulation of the SAPK Hog1, Antifungal drugs such as caspofungin and chemicals such as which in turn mediates the activation of target genes including those Calcofluor White and Congo Red are often used to exert stress upon encoding glycerol biosynthetic enzymes (Smith et al., 2004; the cell wall of C. albicans in vitro (Wiederhold et al., 2005; Eisman The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 et al., 2006; Walker et al., 2008; Leach et al., 2012a). Caspofungin attenuates the induction of these genes, rendering C. albicans and Congo Red interfere with β-glucan synthesis and assembly, sensitive to oxidative stress (Alarco and Raymond, 1999; Enjalbert whereas Calcofluor White perturbs chitin assembly. The cell wall et al., 2006). The redox status of Cap1, and hence oxidative stress changes that occur in response to these artificial insults presumably adaptation, is modulated by the redox regulator thioredoxin (Trx1) reflect normal cell wall homeostasis during growth and development (da Silva Dantas et al., 2010). in the wild, as well as cell wall remodelling events that occur in The Hog1 MAPK pathway also contributes to oxidative stress response to stresses encountered during host–fungus interactions. resistance in C. albicans (Fig. 1). Inactivation of Hog1 and key The Hog1 pathway contributes to cell wall functionality and upstream regulators confer oxidative stress sensitivity (Alonso- regulates chitin biosynthetic functions (Eisman et al., 2006; Munro Monge et al., 2003; Chauhan et al., 2003; Smith et al., 2004; Kruppa et al., 2007). Two additional MAPK pathways contribute to cell wall and Calderone, 2006; da Silva Dantas et al., 2010; Smith et al., stress resistance in C. albicans: the cell integrity pathway (defined 2010). Oxidative stress signals appear to be transduced to Hog1 via by the MAPK Mkc1) and a second pathway that was originally the histidine kinases (Sln1, Chk1, Nik1), the response regulator Ssk1 characterised on the basis of its involvement in yeast-hypha and the peroxiredoxin Tsa1 (Kruppa and Calderone, 2006; Smith et morphogenesis (defined by the MAPK Cek1) (Fig. 1). Both al., 2010). An additional response regulator (Crr1) contributes to pathways are evolutionarily conserved in other fungi (Román et al., oxidative stress resistance in C. albicans, but is not required for 2007). Hog1 activation in response to H O (Bruce et al., 2011). The 2 2 The cell integrity pathway includes a MAPKK module that downstream molecular mechanisms that underlie Hog1-mediated incorporates the MAPKKK Bck1, the MAPKK Mkk1 and the oxidative stress resistance remain an area of active research. The MAPK Mkc1 (Alonso-Monge et al., 2006). Mkc1 activation by cell nuclear accumulation of Cap1 is not dependent on Hog1, and most wall stress is mediated through protein kinase C (Pkc1) signalling oxidative stress-induced transcripts are induced in a Hog1- (Paravicini et al., 1996; Alonso-Monge et al., 2006). The disruption independent fashion (Enjalbert et al., 2006). of Mkc1 confers sensitivity to cell wall stresses and elevated Numerous observations indicate that C. albicans cells are exposed temperatures (Navarro-García et al., 1995). Mkc1 inactivation does to oxidative stress during infection and that oxidative stress not increase the sensitivity of C. albicans to killing by neutrophils adaptation is essential for pathogenicity. There has been or macrophages (Arana et al., 2007), but does attenuate the virulence evolutionary expansion of the SOD gene family in C. albicans, with of C. albicans (Diez-Orejas et al., 1997). this pathogen carrying six superoxide dismutase genes. Transcript The morphogenetic MAPK (Cek1) pathway includes the profiling experiments have demonstrated that oxidative stress genes MAPKKK Ste11, the MAPKK Hst7 and the MAPK Cek1 (Brown, are induced following exposure to host macrophages and neutrophils 2002; Alonso-Monge et al., 2006). Components of this MAPK (Rubin-Bejerano et al., 2003; Lorenz et al., 2004; Fradin et al., module are also involved in the C. albicans mating response (Chen 2005), and during mucosal infection (Zakikhany et al., 2007), but et al., 2002), but Cek2 acts as the MAPK under these conditions. are not activated to the same extent during tissue infection (Thewes The Cek1 pathway is activated via the cell surface sensor Msb2 in et al., 2007; Walker et al., 2009; Wilson et al., 2009). These response to cell wall damaging agents and mutations that affect cell expression patterns have been confirmed by single cell profiling of wall integrity (Román et al., 2009; Cantero and Ernst, 2011). C. albicans cells tagged with diagnostic GFP fusions to oxidative Inactivation of components on the Cek1 pathway inhibits stress genes (Enjalbert et al., 2007; Arana et al., 2007; Miramón et filamentous growth under certain conditions and confers sensitivity al., 2012). The inactivation of genes involved in ROS detoxification, to cell wall stresses (Leberer et al., 1996; Csank et al., 1998; Eisman such as superoxide dismutates and catalase, renders C. albicans cells et al., 2006; Cantero and Ernst, 2011). Candida albicans cek1 more sensitive to phagocytic killing and attenuates the virulence of mutants are not hypersensitive to macrophage or neutrophil killing, the fungus (Wysong et al., 1998; Hwang et al., 2002; Fradin et al., but do display attenuated virulence (Csank et al., 1998; Arana et al., 2005; Frohner et al., 2009). Similar phenotypes are also observed 2007). following the perturbation of oxidative stress regulators. Candida albicans cap1 and hog1 mutants are killed more effectively by Oxidative stress phagocytes (Fradin et al., 2005; Arana et al., 2007), and hog1 and Candida albicans is relatively resistant to reactive oxygen species trx1 mutants display attenuated virulence (Alonso-Monge et al., −1 (ROS), tolerating over 20 mmol l hydrogen peroxide (H O ) under 1999; da Silva Dantas et al., 2010; Cheetham et al., 2011). Taken 2 2 some conditions (Jamieson et al., 1996; Nikolaou et al., 2009; together, the data suggest that C. albicans exploits robust oxidative Rodaki et al., 2009). This resistance is dependent on the AP-1-like stress responses to protect itself from phagocytic killing, but these transcription factor Cap1, which is an orthologue of S. cerevisiae responses become less vital as the fungus develops systemic Yap1 and S. pombe Pap1 (Alarco and Raymond, 1999), and upon infections. the response regulator Skn7 (Singh et al., 2004) (Fig. 1). Cap1 contains redox-sensitive cysteine residues near its carboxy terminus Nitrosative stress that become oxidised following oxidative stress. This leads to the Exposure to reactive nitrogen species (RNS), for example nitric Hog1-independent nuclear accumulation of Cap1 and the activation oxide, causes molecular damage such as the S-nitrosylation of the of its target genes via Yap1-responsive elements (YRE) in their thiol groups of cysteines in proteins and glutathione. RNS exert promoters (Zhang et al., 2000; Enjalbert et al., 2006; Znaidi et al., static rather than cidal effects upon C. albicans (Kaloriti et al., 2009). Cap1 targets include genes involved in the detoxification of 2012). Candida albicans responds to nitrosative stress by activating oxidative stress (e.g. catalase and superoxide dismutase: CAT1 and a defined set of genes that includes oxidative stress functions such SOD1), glutathione synthesis (e.g. gamma-glutamylcysteine as catalase (Cat1), glutathione-conjugating and -modifying enzymes, synthetase: GCS1), redox homeostasis and oxidative damage repair and NADPH oxidoreductases and dehydrogenases (Hromatka et al., (e.g. glutathione reductase and thioredoxin: GLR1 and TRX1). 2005). In addition, YHB1 expression is strongly induced. YHB1 is Together, these functions detoxify ROS and mediate cellular one of three genes encoding flavohaemoglobin-related proteins in adaptation to stress. Consequently, the inactivation of Cap1 C. albicans: YHB1, YHB4 and YHB5 (Ullmann et al., 2004; The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Tillmann et al., 2011). Of these, only YHB1 is induced in response to the types and intensities of stresses that C. albicans encounters to nitric oxide, and this gene encodes the major nitric oxide during its interactions with the host. Third, these stress responses are dioxygenase responsible for nitric oxide detoxification (Ullmann et intimately linked to the virulence of this pathogen (Brown et al., al., 2004; Hromatka et al., 2005). Following RNS detoxification, 2007; Brown et al., 2012a; Román et al., 2007). redox homeostasis is restored and S-nitrosylated adducts are repaired, allowing C. albicans to resume growth (A.T. and A.J.P.B., Adaptation to sequential stresses unpublished). Almost without exception, all of the above studies on stress Little is known about the signalling pathways that mediate the adaptation in C. albicans have examined the responses of cells to nitrosative stress response in C. albicans, or in other yeasts for that individual stresses following growth on glucose. Yet, as described matter. However, it has been shown that the zinc finger transcription above, this pathogen inhabits diverse, complex and dynamic niches factor Cta4 is responsible for activating YHB1 expression in in the host. In these niches C. albicans will be exposed to multiple response to RNS (Chiranand et al., 2008), and the inactivation of stresses. At times these stresses may be imposed sequentially. At either CTA4 or YHB1 confers nitrosative stress sensitivity (Ullmann other times, multiple stresses are imposed simultaneously such that et al., 2004; Chiranand et al., 2008) (Fig. 1). the fungus is exposed to ‘combinatorial stress’. Furthermore, as In addition to ROS, the molecular armoury of phagocytic cells glucose is either limiting or absent from many host niches, C. includes RNS that contribute to fungal killing (Rementería et al., albicans cells must adapt to these stresses whilst exploiting 1995; Vázquez-Torres and Balish, 1997; Brown, 2011). Not alternative carbon sources. Recent data have revealed that these surprisingly, therefore, nitrosative stress genes are induced following factors significantly influence stress adaptation in C. albicans. This phagocytic attack (Fradin et al., 2005; Zakikhany et al., 2007). section addresses adaptation to sequential stresses, and the following Nitrosative stress genes are also upregulated during mucosal section discusses the impact of combinatorial inputs upon stress infections (Zakikhany et al., 2007). However, the response is not adaptation. strongly activated during systemic infection (Thewes et al., 2007; With regard to sequential stresses, it has been known for some Walker et al., 2009), and the inactivation of Yhb1 or Cta4 only time that prior exposure to a non-lethal dose of a stress can protect causes a slight reduction in virulence in the mouse model of yeast cells against a subsequent dose of that same stress (Fig. 2A). systemic candidiasis (Hromatka et al., 2005; Chiranand et al., 2008). For example, acquired thermotolerance has been described in S. Therefore, the nitrosative stress response seems to be most important cerevisiae, S. pombe and more recently in C. albicans (De Virgilio during the early stages of infection when the fungus is battling with et al., 1990; Piper, 1993; Argüelles, 1997). Acquired tolerance has host immune defences. also been observed for oxidative stress in C. albicans (Jamieson et Several common themes are apparent from this brief overview of al., 1996). Acquired stress tolerance is dependent upon the activation key stress responses in C. albicans. First, these stress-signalling of a molecular response to the initial stress, which represents the pathways include regulators that have been highly conserved during induction and accumulation of key proteins or metabolites that fungal evolution. Examples include the Hog1, Mkc1 and Cek1 mediate adaptation to that stress. These proteins and metabolites MAPK modules, and the transcription factors Hsf1 and Cap1. represent a ‘molecular memory’ that can then protect the cell against Second, in comparison with the benign model yeasts S. cerevisiae a subsequent stress, leading to increased survival. However, this and S. pombe, these stress responses have been evolutionarily tuned molecular memory is transient (Leach et al., 2012c), with the length A B Stress Stress Stress 1 Stress 2 Response Response Memory Memory Survival Survival Stress Stress Stress 1 Stress 3 Response Response Memory Memory Survival Survival Time Time Fig. 2. Acquired stress tolerance and stress cross-protection in yeasts. (A) Prior exposure to a stress can protect C. albicans cells against subsequent exposure to that stress (acquired stress tolerance) (upper panel). This indicates the existence of a molecular memory (see ‘Adaptation to sequential stresses’). However, the molecules that represent this memory have biological half-lives. Therefore, this molecular memory is transient, and will be lost during protracted time intervals between stresses (lower panel). (B) In some yeasts, some stresses (stress 1; blue) activate a core transcriptional response (purple) that includes genes that protect against another stress (stress 2; red). In this case, prior exposure to stress 1 often activates a molecular memory that confers protection against stress 2 (upper panel). However, if this core transcriptional response does not include genes that protect against a third stress (stress 3; green), then prior exposure to stress 1 does not activate a relevant molecular memory and does not confer protection against stress 3 (lower panel). The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 of the memory depending upon the decay rates of these proteins and lies much closer to S. cerevisiae than to S. pombe or C. albicans metabolites (Fig. 2A). For example, in the case of thermotolerance, (Roetzer et al., 2008) (Fig. 3). Schizosaccharomyces pombe also the molecular memory in C. albicans probably represents HSPs and displays a core stress response. However, in this case the response is trehalose biosynthetic enzymes rather than the stress protectant driven by Sty1 (Chen et al., 2003), which is the orthologue of the trehalose (Argüelles, 1997; Leach et al., 2012c), because trehalose Hog1 SAPK in S. cerevisiae, C. glabrata and C. albicans (Nikolaou levels decline rapidly once C. albicans cells are returned to lower et al., 2009). In contrast, C. albicans was initially thought to lack a temperatures (Argüelles, 1997). For osmotolerance in C. albicans, core transcriptional response to stress (Enjalbert et al., 2003). the molecular memory is thought to be mediated by glycerol Subsequent work revealed that this yeast does display a core stress biosynthetic enzymes rather than the osmolyte glycerol (You et al., response, but one that comprises a much smaller subset of roughly 25 2012), because glycerol is rapidly extruded from yeast cells when genes (Enjalbert et al., 2006). In C. albicans the roles of Msn2/4-like the osmotic stress is removed (Klipp et al., 2005). By analogy, transcription factors have diverged significantly (Nicholls et al., 2004; acquired tolerance to oxidative stress is probably mediated by the Ramsdale et al., 2008), and the core stress response is coordinated by accumulation of antioxidant enzymes rather than antioxidants Hog1 and Cap1 (Enjalbert et al., 2006). Clearly there has been themselves (Jamieson et al., 1996). significant rewiring of the circuitry that regulates the core stress In some cases, prior exposure to a non-lethal dose of one type of response, as well as of the response itself. stress can also protect yeast cells against a subsequent dose of a This has significant implications for the behaviour of C. albicans different type of stress – a phenomenon called stress cross-protection during exposure to sequential stresses. Thermal stress protects C. (Fig. 2B). For example in S. cerevisiae, a mild heat shock protects albicans against a subsequent oxidative stress, but not against a cells against a subsequent oxidative stress (Wieser et al., 1991; subsequent osmotic or cell wall stress (Enjalbert et al., 2003; Leach Lewis et al., 1995). Similarly, pre-treatment with an oxidative, et al., 2012a). This cross-protection is dependent on Cap1 and osmotic or thermal stress promotes freeze–thaw tolerance in S. correlates with the induction of some Cap1 target genes during heat cerevisiae (Park et al., 1997). The molecular basis for this shock (Nicholls et al., 2009; Leach et al., 2012a). However, this phenomenon lies in the core transcriptional response to stress cross-protection is asymmetric, as an initial treatment with oxidative whereby exposure to any one of several different types of stress stress does not protect C. albicans cells against a subsequent thermal activates genes involved in adaptive responses to many types of stress (Enjalbert et al., 2003; Leach et al., 2012a). stress (Fig. 3). For example, in S. cerevisiae exposure to thermal, These observations are reminiscent of the phenomenon of osmotic, oxidative or pH stress activates several hundred genes with microbial adaptive prediction (Mitchell et al., 2009) (Fig. 4A). roles in stress adaptation, central metabolism and energy generation Mitchell and co-workers argue that some microorganisms inhabit (Gasch et al., 2000; Causton et al., 2001). This core stress response relatively predictable environments, in which one type of is largely dependent on the functionally redundant transcriptional environmental change is often followed by a second type of activators Msn2 and Msn4, which bind to stress response elements stimulus. In such cases organisms may have evolved a regulatory in the promoters of their target genes to mediate their activation circuitry that allows them to predict the second stimulus, thereby (Mager and De Kruijff, 1995; Gasch et al., 2000; Causton et al., conferring an evolutionary advantage. This type of adaptive 2001). Msn2 and its stress-induced transcriptional activation are prediction is displayed by S. cerevisiae, which exploits the elevated downregulated by glucose via the cAMP-protein kinase A (PKA) temperatures associated with vigorous fermentation to induce signalling pathway (Görner et al., 1998; Garreau et al., 2000). oxidative stress genes that will be required once glucose is An analogous Msn2-dependent core transcriptional response to exhausted and cells switch to respiratory and oxidative metabolism stress is displayed by Candida glabrata, which in evolutionary terms (Mitchell et al., 2009). This adaptive prediction is asymmetric, as >400 MYA ~150 MYA ~60 MYA S. cerevisiae C. glabrata C. albicans S. pombe Oxidative Osmotic Oxidative Osmotic Oxidative Osmotic Oxidative Osmotic Glucose Heavy Heat pH Heat Heat starvation Heavy metal metal Msn2/4 Msn2/4 Sty1 CSR CSR CSR CSR ~220 genes ~400 genes ~20 genes ~140 genes Fig. 3. Core stress responses in yeasts. The yeasts C. albicans, Saccharomyces cerevisiae, Candida glabrata and Schizosaccharomyces pombe are evolutionarily separated by many millions of years and occupy contrasting niches: green, environmental niches; red, pathogens. Three of these yeasts display core transcriptional responses to stress in which relatively large numbers of genes are commonly induced in response to different stresses. In S. cerevisiae and C. glabrata the zinc finger transcription factors Msn2 and Msn4 contribute significantly to the core stress response, whereas this response in S. pombe is driven by the Sty1 SAPK. The core transcriptional response has diverged significantly in C. albicans, in which there is a relatively small number of core stress genes (see ‘Adaptation to sequential stresses’). The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Stress 1 Stress 2 Stress 1 Stress 2 Response 1 Response 2 Response 1 Response 2 Asymmetric Symmetric adaptive prediction adaptive prediction Elevated Oxidative Elevated Oxidative Glucose temperature stress temperature stress exposure HSP Oxidative HSP Oxidative Metabolic genes stress genes genes stress genes genes S. cerevisiae C. albicans Fig. 4. Anticipatory prediction in C. albicans and S. cerevisiae. (A) As described by Mitchell and co-workers, microbes often display adaptive prediction, whereby exposure to one environmental input can lead to the anticipatory induction of the response to a second environmental input (Mitchell et al., 2009). The authors argue that this provides an evolutionary advantage to the microbe because the first input is often followed by the second input in its normal environmental niche. Anticipatory responses can be asymmetric or symmetric. (B) Saccharomyces cerevisiae displays asymmetric anticipatory adaptive prediction by activating oxidative stress genes in response to elevated temperatures. Candida albicans displays an analogous asymmetric anticipatory adaptive response (Mitchell et al., 2009). This pathogen also displays symmetric anticipatory adaptive prediction by activating oxidative stress genes in response to glucose exposure and by activating carbohydrate metabolism in response to oxidative stress (see ‘Adaptation to sequential stresses’). oxidative stress does not induce heat shock gene expression in S. Adaptation to combinatorial stresses cerevisiae (Mitchell et al., 2009). An analogous asymmetric As mentioned above, C. albicans cells are often simultaneously relationship between oxidative and heat shock gene regulation is exposed to multiple stresses within the complex host niches they observed in C. albicans: in general, oxidative stress functions are inhabit. Possibly the best example of combinatorial stress occurs induced in response to heat shock, but heat shock genes are not following phagocytosis by neutrophils or macrophages, when the induced by an oxidative stress (Enjalbert et al., 2003) (Fig. 4B). This fungus is bombarded with ROS, RNS and cationic fluxes is consistent with the idea that adaptive prediction might have (Rementería et al., 1995; Vázquez-Torres and Balish, 1997; Brown, evolved in C. albicans such that the pathogen anticipates oxidative 2011; Nüsse, 2011). However, combinatorial stresses are likely to attack by phagocytic cells in response to fevers associated with be relevant in many other host niches, such as during mucosal inflammatory responses. invasion (where oxidative stresses are encountered while adjusting A second example of adaptive prediction has been described in C. cellular water balance) and kidney infection (where respiring cells albicans. In this fungus, oxidative stress genes are activated must deal with endogenous ROS while adapting to relatively high following exposure to glucose, thereby conferring elevated salt concentrations). How do C. albicans cells respond to such resistance to acute oxidative stress (Rodaki et al., 2009) (Fig. 4B). combinatorial stresses? We have predicted that the adaptive This phenomenon does not depend on Hog1 or Cap1 (Rodaki et al., responses to such combinatorial stresses might not be equivalent to 2009). Instead, glucose-enhanced oxidative stress resistance appears the sum of the responses to the corresponding individual stresses to be regulated by evolutionarily conserved glucose signalling (Kaloriti et al., 2012). Our rationale is that unexpected cross-talk pathways (I.B. and A.J.P.B., unpublished). This anticipatory between the relevant signalling pathways might exist. Several response, which is triggered by the glucose concentrations present examples of this have emerged recently. in the bloodstream, is likely to be relevant in the disease context. Combinatorial oxidative (H O ) plus nitrosative stresses 2 2 Candida albicans cells that enter the bloodstream are exposed to (dipropylenetriamine-NONOate, DPTA-NONOate) and glucose, and this may help to protect them against the impending combinatorial cationic (NaCl) plus nitrosative stresses appear to attack from phagocytic cells. If this were true, the phenomenon of exert additive effects upon the growth of C. albicans cells (Kaloriti glucose-enhanced oxidative stress resistance must have evolved et al., 2012). However, YHB1 gene induction is attenuated under relatively recently. This appears to be the case (I.B. and A.J.P.B., these conditions, indicating that Cta4 signalling is compromised unpublished). Indeed, the opposite phenotype is observed in S. (A.T. and A.J.P.B., unpublished). Significantly, non-additive effects cerevisiae: glucose reduces stress resistance in this benign yeast, are observed for combinatorial cationic plus oxidative stresses which has evolved in environmental niches (Mager and De Kruijff, (Kaloriti et al., 2012). These stresses kill C. albicans synergistically. 1995; Görner et al., 1998; Garreau et al., 2000). The basis for this appears to be ‘stress pathway interference’, This anticipatory response appears to be symmetric because whereby both Cap1 and Hog1 signalling are compromised by the exposing C. albicans cells to hydrogen peroxide leads to the combination of cationic and oxidative stress. As a result, cationic activation of genes involved in central carbon metabolism (Enjalbert and oxidative stress genes are not induced, and intracellular ROS et al., 2006). However, this particular response (oxidative stress- levels increase, leading to cell death (D.K., M.D.J., A.T. and induced metabolic activation), which is conserved in other yeasts A.J.P.B., unpublished). Indeed, hydrogen peroxide has been shown (Gasch et al., 2000; Causton et al., 2001; Chen et al., 2003; Enjalbert to stimulate apoptotic cell death in C. albicans via Ras-cAMP et al., 2006), may have less to do with anticipatory prediction and signalling (Phillips et al., 2003; Phillips et al., 2006). This appears more to do with the need for metabolic intermediates and energy to to be highly relevant to host–fungus interactions because the drive oxidative stress adaptation (Brown et al., 2012a). effective killing of C. albicans cells by human neutrophils appears The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 to depend on the extreme potency of combinatorial cationic plus (reviewed by Brown et al., 2009). We have now shown that oxidative stresses (D.K., M.D.J., A.T. and A.J.P.B., unpublished). combinatorial effects can also be triggered at the biochemical level. Combinatorial effects are also observed between thermal and In this case the inhibition of key detoxification functions by cationic other stresses. For example, elevated temperatures decrease the stresses leads to the build up of intracellular ROS, causing stress sensitivity of C. albicans cells to a cell wall stress (Calcofluor pathway interference and ultimately cell death (D.K., M.D.J., A.T. White), but have little effect upon osmo-sensitivity (Leach et al., and A.J.P.B., unpublished). In addition, we have shown that 2012a). This stress interaction appears to be mediated via Hsp90 combinatorial effects can be mediated by a biological transistor. In (Leach et al., 2012a). As described above, the Hog1, Mkc1 and this case, Hsp90 coordinates the activities of multiple signalling Cek1 pathways modulate cell wall functionality. The MAP kinases pathways involved in cellular adaptation (Leach et al., 2012b). in these pathways are all client proteins of Hsp90, and their While the responses of fungal cells to individual stresses are now activation is modulated by Hsp90 (Leach et al., 2012a). Temperature reasonably well understood, little is known about the mechanisms fluctuations have been shown to influence HSP90 expression levels that underlie combinatorial stress adaptation. Yet, combinatorial as well as the binding of Hsp90 to its client proteins in C. albicans stress adaptation is highly relevant to natural environments. (Nicholls et al., 2009; Diezmann et al., 2012; Leach et al., 2012a; Leach et al., 2012c). Furthermore, ambient temperature affects the Impact of dynamic host niches upon stress adaptation cell wall proteome, and Hsp90 depletion alters cell wall architecture Metabolic changes within host niches also affect stress adaptation in (Leach et al., 2012a; Heilmann et al., 2013). Therefore, Hsp90 has C. albicans (Fig. 6). In particular, many host niches either lack been proposed to act as a biological transistor that tunes sugars such as glucose or contain glucose at low concentrations. environmental responses, including cell wall remodelling, to the Instead, these niches contain complex mixtures of alternative carbon ambient temperature of the cell (Leach et al., 2012b). sources such as amino acids, carboxylic acids such as lactate, and Therefore, as predicted (Kaloriti et al., 2012), combinatorial fatty acids. Consequently, C. albicans must assimilate these stresses exert unexpected effects upon the classical regulatory alternative carbon sources if it is to grow and colonise these niches. pathways that mediate responses to specific stresses (Fig. 1). The Not surprisingly, metabolic pathways that are essential for the available data have revealed several distinct molecular mechanisms assimilation of these alternative carbon sources, such as by which combinatorial cross-talk can occur (Fig. 5). First, there gluconeogenesis and the glyoxylate cycle, are required for full appears to be signalling cross-talk between the MAPKs in critical virulence (Lorenz and Fink, 2001; Barelle et al., 2006; Piekarska et stress signalling pathways. This is suggested by mutational analyses al., 2006; Ramírez and Lorenz, 2007). Furthermore, lactate whereby the deletion of HOG1 leads to the derepression of Cek1 assimilation is essential for C. glabrata to colonise the intestine phosphorylation and the inhibition of Mkc1 phosphorylation (Arana (Ueno et al., 2011), and a significant proportion of C. albicans cells et al., 2005). Cross-talk also exists at the chemical level. infecting the kidney activate pathways for alternative carbon Combinations of H O and NaCl lead to the formation of utilisation (Barelle et al., 2006), as do phagocytosed C. albicans 2 2 hypochlorous acid (HOCl), and nitric oxide and superoxide react to cells (Lorenz et al., 2004; Fradin et al., 2005; Barelle et al., 2006; form peroxynitrite (ONOO ), and nitrite and hypochlorous acid Miramón et al., 2012). The metabolic activity of C. albicans can combine to form nitryl chloride (NO Cl), generating cocktails of modify the pH of its microenvironment (Vylkova et al., 2011) toxic compounds that can damage lipids, proteins and nucleic acids adding to the dynamism of host niches. The regulatory circuitry that Temperature A B Bck1 Ste11 Ssk2 Hsp90 Hst7 Pbs2 Mkk1 Hsf1 Cek1 Hog1 Mck1 Hog1 Cek1 Mck1 Thermotolerance Cationic Oxidative C D plus stress stress superoxide H O 2 2 NO ONOO hydrogen nitric peroxide peroxynitrite HOCl oxide Cationic Oxidative hypochlorous stress genes stress genes acid RCS ROS RNS Stress genes Fig. 5. Mechanisms underlying combinatorial stress effects in C. albicans. Several distinct mechanisms contribute to combinatorial stress effects in C. albicans (see ‘Adaptation to combinatorial stresses’). (A) Classical cross-talk occurs between the MAPK signalling pathways (Alonso Monge et al., 2006). Hog1 signalling pathway: Ssk2, MAPKKK; Pbs2, MAPKK; Hog1, MAPK/SAPK. Cell integrity pathway: Bck1, MAPKKK; Mkk1, MAPKK; Mkc1, MAPK. Mating/invasive growth pathway: Ste11, MAPKKK; Hst7, MAPKK; Cek1, MAPK. (B) Hsp90 acts as a biological transistor, modulating the activities of the transcription factor Hsf1 and the MAPKs in response to thermal fluctuations (Leach et al., 2012a; Leach et al., 2012b). (C) Combinatorial cationic plus oxidative stress leads to stress pathway interference, whereby Hog1 and Cap1 signalling are affected by oxidative and cationic stress, respectively (D.K., M.D.J., A.T. and A.J.P.B., unpublished). (D) There is cross-talk at the chemical level, whereby different reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive chlorine species (RCS) can be generated spontaneously and by enzymatic catalysis (Brown et al., 2009; Brown et al., 2011), presumably leading to the activation of different subsets of stress genes. The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Carbon source Proteome Architecture Cell wall Biophysical properties Glucose Lactate Stress Immune adaptation recognition Virulence Fig. 6. Impact of carbon source on C. albicans. Changes in carbon source affect the proteome, architecture and biophysical properties of the C. albicans cell wall. This affects stress adaptation, immune recognition and virulence (Ene et al., 2012a; Ene et al., 2012b; Ene et al., 2013). Transmission electron micrographs of cell walls from C. albicans cells grown on glucose or lactate are shown on the right. regulates carbon assimilation in C. albicans has undergone individual cells vary even within specific host niches. Therefore, the evolutionary rewiring (Ihmels et al., 2005; Martchenko et al., 2007; spatial regulation of stress adaptation must also be examined during Lavoie et al., 2009; Sandai et al., 2012), just as is the case for stress infection. This must either be done by examining the responses of adaptation (discussed above). individual cells in vivo, for example using GFP-based single-cell Despite the fact that glucose is limiting or absent in many host profiling methods (Barelle et al., 2006; Enjalbert et al., 2007; niches, most studies of stress adaptation in C. albicans have been Miramón et al., 2012), or by increasing the sensitivity of RNA performed on cells grown in media containing 2% glucose. sequencing technologies and increasing their spatial resolution, for Recently, we showed that growth on physiologically relevant example by exploiting laser capture microscopy. These approaches alternative carbon sources, such as lactate or oleic acid, affects stress are being pursued by the Aberdeen Fungal Group (J.P., S.S. and adaptation in C. albicans (Ene et al., 2012a). Lactate-grown cells are A.J.P.B., unpublished). more resistant to osmotic stress, cell wall stresses and some In addition, at least three aspects of stress adaptation that are of antifungal drugs. This increased stress resistance is not dependent direct relevance in vivo need further dissection in vitro. First, which on Hog1 or Mkc1 signalling. Instead, it relates to the effects of anticipatory responses in C. albicans influence host colonisation and alternative carbon sources on the proteomic content and architecture disease progression, and how are these anticipatory responses of the cell wall, which in turn impact upon the biophysical properties controlled at the molecular level? Second, which combinatorial of the cell wall (Ene et al., 2012a; Ene et al., 2012b) (Fig. 6). These stress responses in C. albicans influence host–fungus interactions, alterations at the cell surface affect host recognition of C. albicans and how are they regulated? Third, how does metabolic adaptation cells and influence the virulence of this pathogen in both systemic influence stress resistance within host niches? Despite the limited and mucosal models of infection (Ene et al., 2012a; Ene et al., exploration of these issues, it is already clear that they involve non- 2013). Clearly, metabolic adaptation affects stress responses in C. additive behaviours that reflect unexpected signalling, albicans, and this further complicates our understanding of transcriptional, biochemical and chemical cross-talk. Furthermore environmental adaptation of this fungus within the complex and many of these responses are dynamic and dose dependent. Given dynamic microenvironments it occupies during host colonisation their complexity, a combination of experimental approaches and and disease progression. Significantly, this is also likely to affect the predictive mathematical modelling seems especially important for efficacy of antifungal drug treatments against individual C. albicans the development of a true understanding of these adaptive processes. cells in these niches (Ene et al., 2012a). Such studies will provide important insights into the forces that have driven the recent evolution of this pathogen in its host. Outlook In closing, it is worth emphasising that studies of stress adaptation Significant advances have been made in our understanding of stress are revealing points of fragility in C. albicans that could potentially adaptation in C. albicans, and progress is being made towards the provide targets for translational research directed towards the elaboration of specific stress signalling pathways. This is important development of novel antifungal therapies. Indeed, the therapeutic because stress adaptation contributes to the virulence of this major potential of Hsp90 inhibitors is being pursued by a number of fungal pathogen of humans. However, host niches are complex and laboratories (Dolgin and Motluk, 2011). Therefore, observations dynamic, and the impact of this complexity and dynamism upon such as the acute sensitivity of C. albicans towards combinatorial stress adaptation remains largely unexplored. In particular, how are cationic plus oxidative stress could, in principle, be exploited stress responses regulated temporally during host colonisation and therapeutically. disease progression? The elegant microarray studies performed by Bernie Hube’s group go some of the way to addressing this question Acknowledgements We thank our friends and colleagues in the Aberdeen Fungal Group, the CRISP (Fradin et al., 2005; Thewes et al., 2007; Zakikhany et al., 2007; Consortium, the FINSysB Network and the Cowen laboratory for stimulating Wilson et al., 2009). However, microarray studies average the discussions and helpful advice. Neil Gow, Frank Odds, Carol Munro, Gordon molecular behaviour of the fungal population as a whole, and fungal Brown, Janet Quinn, Ken Haynes, Christophe d’Enfert, Bernard Hube, Mihai populations display heterogeneous behaviours in host niches Netea, Frans Klis, Leah Cowen, Stephanie Diezmann and Joe Heitman deserve (Barelle et al., 2006). This is because the microenvironments of special mention. The Journal of Experimental Biology REVIEW The Journal of Experimental Biology (2014) doi:10.1242/jeb.088930 Competing interests of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459, 657-662. The authors declare no competing financial interests. Calderone, R. (2002). Candida and Candidiasis. Washington, DC: ASM Press. Calderone, R. A. and Clancy, C. J. (2012). Candida and Candidiasis, 2nd edn. Author contributions Washington, DC: ASM Press. All authors contributed to the writing of this review, the initial draft being prepared Cantero, P. D. and Ernst, J. F. (2011). Damage to the glycoshield activates PMT- by A.J.P.B. and M.D.L. directed O-mannosylation via the Msb2-Cek1 pathway in Candida albicans. Mol. Microbiol. 80, 715-725. Funding Causton, H. C., Ren, B., Koh, S. S., Harbison, C. T., Kanin, E., Jennings, E. G., Lee, T. I., True, H. L., Lander, E. S. and Young, R. A. (2001). Remodeling of yeast We are grateful to our funding bodies for their support. This work was supported by genome expression in response to environmental changes. Mol. Biol. Cell 12, 323- the European Commission [FINSysB, PITN-GA-2008-214004; STRIFE, ERC- 2009-AdG-249793], by the UK Biotechnology and Biological Research Council Chauhan, N., Inglis, D., Roman, E., Pla, J., Li, D., Calera, J. A. and Calderone, R. [grant numbers BBS/B/06679; BB/C510391/1; BB/D009308/1; BB/F000111/1; (2003). Candida albicans response regulator gene SSK1 regulates a subset of BB/F010826/1; BB/F00513X/1], and by the Wellcome Trust [grant numbers genes whose functions are associated with cell wall biosynthesis and adaptation to 080088, 097377]. M.D.L. was also supported by a Carnegie/Caledonian oxidative stress. Eukaryot. Cell 2, 1018-1024. Scholarship and a Sir Henry Wellcome Postdoctoral Fellowship from the Wellcome Chauhan, N., Latge, J. P. and Calderone, R. A. (2006). Signalling and oxidant Trust [grant number 096072]. 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Journal of Experimental Biology – The Company of Biologists

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