TY - JOUR AU1 - Rehman, Saima AU2 - Grigoryeva, Lubov S. AU3 - Richardson, Katherine H. AU4 - Corsini, Paula AU5 - White, Richard C. AU6 - Shaw, Rosie AU7 - Portlock, Theo J. AU8 - Dorgan, Benjamin AU9 - Zanjani, Zeinab S. AU1 - Fornili, Arianna AU1 - Cianciotto, Nicholas P. AU1 - Garnett, James A. AB - Introduction Legionella pneumophila is a Gram-negative bacterium that can withstand large variation in pH and temperature. When humans are exposed to L. pneumophila, it can infect macrophages and epithelia in the lungs and trigger chronic inflammation and tissue damage [1]. L. pneumophila is the causative agent of Legionnaires’ disease, an often-fatal pneumonia, and Pontiac fever, a milder flu-like disease, and rates of infection are increasing each year [1–4]. Although infection is primarily via inhalation of contaminated water droplets from aerosolizing devices [5], there is also now evidence for person-to-person transmission [6, 7]. Upon invasion of eukaryotic hosts, L. pneumophila avoids fusion with canonical endosomal/lysosomal pathways by forming a membrane bound compartment, the Legionella containing vacuole (LCV) [1]. L. pneumophila exports over 300 proteins from this modified phagosome into the host cytoplasm by the Icm/Dot type IVb secretion system [1]. These effectors manipulate host signalling pathways and mediate evasion of the host’s degradative lysosomal pathway, enabling L. pneumophila to replicate to large numbers [8]. L. pneumophila also expresses a type II secretion system (T2SS) [9], which secretes at least 25 proteins, including almost 20 enzymes and substrates that contain a high proportion of unique amino acid sequence with no homology outside of the Legionella genus [10, 11]. The T2SS is important for both intracellular and extracellular lifestyles [10]. These processes include extracellular growth at low temperatures, biofilm formation, intracellular replication in amoebae and macrophages, dampening of cytokine output from infected cells and persistence in lungs [10, 12–18]. Among the L. pneumophila type II substrates, ChiA is an 81 kDa endochitinase with a novel amino acid sequence at its N-terminus and a putative glycosyl hydrolase 18 (GH18) domain at its C-terminus [17]. Chitin is an insoluble carbohydrate composed of linear β-1,4-linked N-acetylglucosamine (GlcNAc) residues and its degradation by chitinase enzymes serves as an important source of nutrients for many bacteria [19]. Chitin is not synthesized by mammals and L. pneumophila chiA mutants are not impaired for growth in Acanthamoeba castellanii, Vermamoeba vermiformis, and Willaertia magna amoebae, macrophage-like U937 cells, and lung epithelial cell-like A549 cells [14, 17, 20, 21]. However, they are less able to survive in the lungs of A/J mice, suggesting that ChiA is required for optimal survival of L. pneumophila in the lungs [17], although how it is able to promote infection is unknown. ChiA is present within 53% of Legionella species, and its closest homologs outside of the Legionella genus are within other γ-Proteobacteria, especially species of Aquicella that, like Legionella, infect amoebae [10]. Interestingly, L. pneumophila ChiA also has high relatedness to proteins encoded by both mimiviruses that infect amoebae and water moulds, raising the possibility that ChiA was acquired by inter-kingdom horizontal gene transfer [10]. In this study, we report a structural model for full-length ChiA based on X-ray crystallographic, template based modelling and small angle X-ray scattering (SAXS) data. ChiA is composed of four domains (N1, N2, N3 and CTD, from N- to C-terminus) which have structural homology to those associated with other chitinase enzymes. Using chitin binding and chitinase activity assays, we show that ChiA-N1 is a chitin binding module and we confirm that ChiA-CTD is a glycosyl hydrolase domain. Using binding assays, we show that both the ChiA-N3 domain and eukaryotic mucins can associate with the L. pneumophila surface. Lastly, our structural and biochemical studies demonstrate that ChiA-CTD has novel peptidase activity against mucin-like glycoproteins, which is independent of its chitinase active site. Our work provides novel molecular insight into the virulence mechanism of a bacterial chitinase and suggests that ChiA has a role in modulating host immune responses and facilitates L. pneumophila penetration of the alveolar mucosa during infection. Results ChiA is a multi-domain protein Full-length ChiA from L. pneumophila 130b (ChiA-FL; numbered 1–762 for the mature protein; NCBI accession WP_072401826.1) with an N-terminal His6-tag was produced in Escherichia coli K12 and purified by nickel-affinity and size exclusion chromatography. Despite extensive screening ChiA-FL resisted crystallization and we therefore used bioinformatics analysis to produce a series of subdomain constructs for further characterization (Fig 1A). While previous examination of the C-terminal domain of ChiA (ChiA-CTD: residues 419–762) had revealed high primary sequence homology to other GH18 chitinase domains [17], the N-terminal region (ChiA-NT; residues 1–417) contains unique primary sequence with no significant homology to any other known protein. Nonetheless, through secondary structure prediction [22] and template based modelling using the Phyre2 [23] and Robetta [24] servers, we identified three putative N-terminal subdomains based on predicted structural similarity with carbohydrate-binding modules (CBMs; ChiA-N1: residues 1–140), fibronectin type-III-like domains (Fn3; ChiA-N2: residues 138–299) and a chitinase A N-terminal domain (ChiN; ChiA-N3: residues 285–417) (Fig 1A and S1 Table). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Chitin binding and endochitinase functions of ChiA. (A) Schematic representation of ChiA with domain boundaries annotated. (B) ChiA-FL, subdomains (NT, N1, N2, N3, CTD) and ChiA-CTD mutants (E543M, Q583A, Q617A) were assayed for chitinase activity against p-NP-[GlcNAc]3. Data represent the mean and standard deviation for triplicate experiments. (C) Chitin pull-down experiment to assess direct interactions between immobilized chitin and ChiA. ChiA-FL and subdomains (NT, N1, N2, N3, CTD, CTDE543M) were incubated with chitin beads and analysed by SDS-PAGE. BSA was used as a control. L: loaded sample; B: eluted beads. Eluted samples undergo an upward shift compared to the input sample due to differences in buffer conditions. Data is representative of three independent repeat experiments. https://doi.org/10.1371/journal.ppat.1008342.g001 To examine the function of the ChiA N-terminal domains we began by examining their endochitinase activity [17]. Each construct was expressed with an N-terminal His6-tag in E. coli K12 and purified by nickel-affinity and size exclusion chromatography. All reagents were well folded as determined by 1D 1H nuclear magnetic resonance (NMR) spectroscopy (S1 Fig). As expected, ChiA-FL and ChiA-CTD were both active against p-nitrophenyl β-D-N,N′,N″ triacetylchitotriose (pNP-[GlcNAc]3) but no activity was detected for ChiA-NT or an E543M ChiA-CTD active site mutant (ChiA-CTDE543M) (Fig 1B and S2 Fig). We then assayed binding of ChiA sub-domains to immobilized chitin and observed that in addition to ChiA-CTD, the N1-domain also recognizes chitin polymers, which supports its role as a carbohydrate binding module (Fig 1C). Atomic structure of ChiA-CTD We next initiated crystallographic studies of the ChiA subdomains. We readily obtained crystals for ChiA-CTD and its structure was determined using iodide single isomorphous replacement with anomalous scattering (I-SIRAS) phasing. Electron-density maps were refined to 1.7 Å (Table 1) and the final model contains two identical chains, with all molecules built except for the N-terminal His6-tags and adjacent ChiA-CTD residues Val419 to Gly424. Each chain forms an anticipated GH18 α/β-fold and is composed of 11 β-strands and 13 α-helices (Fig 2A). High concentrations of 2-methyl-2,4-pentanediol (MPD) were used as a precipitant during crystallization and we observed four MPD molecules in the final model; one bound to the catalytic Asp541 and Glu543 residues (MPD-1) and one within a hydrophobic pocket formed by the α5 helix and α5-α5′/β5-β6′ loops (MPD-2) (S3 Fig). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Crystal structure of ChiA-CTD. (A) Cartoon representation of ChiA-CTD with secondary structure and extended loops annotated. Additional ChiA-CTD α3-helix is highlighted with a red asterisk. (B) Stick representation of the ChiA-CTD active site (green) superimposed with ChiNCTU2 E145G/Y227F mutant (grey) in complex with chitotetraose (yellow) (PDB ID code 3n18) [27]. Mutated residues in ChiNCTU2 are indicated and carbohydrate positions relative to the hydrolysed glycosidic bond are numbered. (C) Model of ChiA-CTD shown as cartoon and (D) electrostatic surface potential, bound to chitotetraose drawn as spheres. https://doi.org/10.1371/journal.ppat.1008342.g002 Download: PPT PowerPoint slide PNG larger image TIFF original image Table 1. Diffraction data and refinement statistics. https://doi.org/10.1371/journal.ppat.1008342.t001 The overall structure of ChiA-CTD is highly similar to other GH18 chitinase domains, and the Dali server [26] identified Bacillus cereus ChiNCTU2 enzyme inactive E145G/Y227F mutant in complex with chitotetraose (Protein Data Bank (PDB) ID code 3n18) [27]; Bacillus anthracis Chi36 (PDB ID code 5kz6); Chromobacterium violaceum ChiA (PDB ID code 4tx8); and Streptomyces coelicolor ChiA (PDB ID code 3ebv) as having the highest homologies (Z score: 36.3, 35.9, 34.4, 34.1 respectively; rmsd: 2.2 Å, 2.3 Å, 2.2 Å, 1.8 Å, respectively). The chitinase active sites of ChiNCTU2 and ChiA-CTD have high primary sequence identity and tertiary structure homology (Fig 2B and S4 Fig) and modelling of chitotetraose binding indicates that chitin lines a negatively charged valley on the surface of ChiA-CTD (Fig 2C and 2D). Chitotetraose overlays with MPD-1 in the active site (S3 Fig) and the MPD-2 site is positioned adjacent to the reducing end of the modelled chitotetraose. In this binding model, Gln583 and Gln617 have a central role in the correct positioning of chitotetraose in the ChiA-CTD active site. We therefore created Q583A and Q617A mutants in recombinant ChiA-CTD and as anticipated, these mutants showed no activity against pNP-[GlcNAc]3 (Fig 1B and S2 Fig). However, L. pneumophila ChiA-CTD also possesses unique features that are not observed in homologous structures. These include an additional α-helix (α3), an extended β3-α3 loop, an extended α6-α6′ loop and an extended β7-α7 loop (Fig 2A and S4 and S5 Figs). ChiA is an elongated and dynamic structure in solution We used small angle X-ray scattering (SAXS) to model the global structure of full-length ChiA in solution. Four different concentrations at 4, 2, 1, and 0.5 mg/ml were measured but signs of aggregation were apparent at concentrations above 1 mg/ml (S6 Fig). To achieve the highest signal/noise ratio, all further analysis was carried out with the data from the 1 mg/ml sample (S6 Fig and S2 Table). Guinier analysis suggested a radius of gyration (Rg), the root mean square distance to the particles centre of mass, of 5.43 nm and analysis of the distance distribution function (P(r)) suggested a maximum particle dimension (Dmax) of 17.77 nm and Rg of 5.45 nm (S6 Fig). Using BSA as a standard, we calculated a particle molecular mass of 89.2 kDa, which is within the method error range for a monomeric 82.6 kDa ChiA. Kratky, Kratky-Debye and Porod-Debye plot analyses of the SAXS data indicated that ChiA is a highly dynamic particle in solution (S6 Fig) [28]. This is likely due to flexibility within the ChiA inter-domain linkers and we therefore used the ensemble optimization method (EOM) to determine molecular model ensembles of ChiA that best fit the SAXS data [29]. As we were not able to obtain crystals for ChiA N-domains, an initial model of ChiA was created using a Phyre2 derived N1-domain (residues 22–147), a Robetta derived N2-domain (encompassing two further subdomains: residues 152–245 and 248–305), a Phyre2 derived ChiA N3-domain (residues 315–414) and the crystal structure of ChiA-CTD (residues 439–777), separated by flexible linkers (Fig 3A and S1 Table) [23, 24, 29]. Ensemble optimization analysis of the scattering data yielded an excellent fit between experimental and calculated SAXS profiles (χ2: 1.1), which again indicates that ChiA is highly flexible in solution (Rflex 91.4) [30] with conformation ensembles clustered within three populations (Fig 3B and 3C and S3 Table). The majority of the simulated conformations exhibited partially extended or fully extended structures at Rg 44–56 Å or Rg 60–71 Å, respectively, whereas minor conformations of closed structures were also populated at Rg 33–43 Å. This analysis provides experiential support for the global features of our ChiA N-domain models but also suggests that there is cooperation between ChiA domains. As inter-domain flexibility is a key feature of processive enzymes [31], these data suggest that processivity may also be important for the function of ChiA. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. Model of ChiA-FL in solution. (A) Initial model of ChiA generated by EOM 2.0 [30]. Linkers are shown as grey spheres. (B) EOM fit (orange line) to the ChiA SAXS data (black open circles) with χ2 of 1.09. (C) Three independent ensemble optimization method runs (yellow, orange and burgundy) yielded similar distributions of three populations. Sample ChiA models corresponding to the centre of each population for all three runs are shown. https://doi.org/10.1371/journal.ppat.1008342.g003 ChiA is targeted to the L. pneumophila surface Although we have repeatedly detected secreted ChiA in bacterial culture supernatants [17, 32], it has been well documented in other bacteria that some type II substrates associate with the bacterial surface upon their secretion [11, 33]. To determine whether ChiA is also targeted to the bacterial surface, L. pneumophila 130b was examined by whole-cell ELISA using anti-ChiA, anti-Mip and anti-ProA antibodies [32, 34] (Fig 4A). Both ChiA and Mip, a known surface exposed protein [35], were positive by ELISA, whilst the metalloprotease ProA, another T2SS substrate [10, 36], was negative. As the translocation signal for ChiA (and T2SS substrates in general) has yet to be determined [10, 11], in vivo truncations or site directed mutagenesis of chiA could inadvertently impede the secretion of ChiA. Therefore, to examine which region of ChiA is responsible for this localization, a L. pneumophila NU318 (chiA) mutant [17] was incubated with recombinant ChiA fragments and the whole-cell ELISA was repeated using ChiA antisera (Fig 4B). ChiA-NT and the N3-domain exhibited significant binding to the bacterial surface while no binding was observed for ChiA-CTD, ChiA-N1 or ChiA-N2. A direct comparison between anti-ChiA antibodies binding to ChiA-FL, ChiA-NT and ChiA-CTD indicated that the antiserum binds to the N- and C-terminal fragments at a similar or higher level than to full length ChiA (S7 Fig). Likewise, direct comparison between the three N-terminal subdomains revealed that anti-ChiA antibodies bind to the N1 and N2 subdomains with comparable or higher affinity as they do to ChiA-N3 (S7 Fig). Thus, the greater association of ChiA-FL, ChiA-NT and ChiA-N3 with the L. pneumophila surface, as detected by whole-cell ELISA (Fig 4B), is not simply the result of a reduced ability of the antiserum to recognize ChiA-CTD, ChiA-N1, and ChiA-N2. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 4. L. pneumophila surface association of ChiA and mucin binding. (A) Whole cell ELISA of L. pneumophila wild-type 130b (WT), chiA mutant NU318 (chiA), proA mutant AA200 (proA) and mip mutant NU203 (mip) detected with antiserum specific for either ChiA, ProA, or Mip. (B) Whole cell ELISA of chiA mutant incubated with either recombinant ChiA-FL or subdomains (NT, N1, N2, N3, CTD) and detected with antiserum specific for ChiA. PBS buffer alone was used as a control (-). Multiple comparisons against the control were made using a one-way ANOVA, Holm-Šídák multiple comparisons test; * P < 0.05, *** P < 0.001. (C) ELISA analysis of binding between immobilised type I-S, II or III porcine stomach mucins and His-tagged ChiA-FL, subdomains (NT, N1, N2, N3, CTD) and controls (SslE, NttE) detected with anti-His-tag antibody. BSA-coated wells were used as controls. *** P < 0.001; verses control empty well by two-tailed Student’s test. (D) Mucin binding to GFP-expressing L. pneumophila WT or chiA mutant strains were incubated at 25°C or 37°C with PBS, type I-S, II or III mucins followed by Texas Red-tagged wheat germ agglutinin (WGA). Mucin binding to bacteria was quantified by flow cytometry. * P < 0.05; verses PBS control by two-tailed Student’s test. All data represent the mean and standard deviation for triplicate experiments. https://doi.org/10.1371/journal.ppat.1008342.g004 ChiA is a mucin binding protein Some bacterial chitinases and chitin binding proteins are able to promote infection through adhesion to and/or degradation of host glycoconjugates [37] and we hypothesized that ChiA may interact with exogenous mucins in the lungs and elsewhere. We therefore examined the binding capacity of recombinant ChiA-FL, ChiA domains, L. pneumophila NttE, another T2SS substrate [10, 17], and E. coli SslE, a known mucin binding protein and mucinase [38], to immobilized commercially available mucin extracts by ELISA using anti-His antibodies (Fig 4C). All ChiA samples and SslE displayed significant adhesion to mucins isolated from porcine stomachs (type II and III), but this was not observed with a mucin extract from bovine submaxillary glands (type I-S). Conversely, NttE showed no binding to any of the mucin samples. This confirmed that ChiA has additional specificity for non-chitinous ligands and implied that ChiA could bind host glycoproteins on the Legionella surface. To assess this, we incubated L. pneumophila 130b wild-type and NU318 (chiA) mutant strains with type I-S, II and III mucin extracts followed by wheat germ agglutinin and measured their binding to the bacterial surface by flow cytometry. Whether examined at 25°C or 37°C, type II and III extracts showed strong association to both L. pneumophila strains, but submaxillary gland mucins did not (Fig 4D). However, there was no significant difference in binding of the type II and III mucins between wild-type and chiA mutant strains, which indicates that other factors apart from ChiA are present on the Legionella surface that can also recognize mucins and in the case of the mutant, compensate for the loss of ChiA. Interestingly, the mean binding of L. pneumophila to both type II and III mucins trends slightly higher in the chiA mutant strain and we speculated that this may be due to ChiA degrading mucins on the bacterial surface on the wild-type strain. ChiA increases penetration of L. pneumophila through the mucin layer We next examined whether secreted ChiA is able to degrade mucins. Porcine stomach type II mucin extract was incubated with supernatants from L. pneumophila 130b wild-type and NU318 (chiA) mutant strains or a cocktail of enzymes (pepsin, pronase, β-N-acetylglucosaminidase, fucosidase) with known activity against mucins [39–41], and then analysed by immunoblotting using wheat germ agglutinin (Fig 5A, left panel). While the majority of the mucin extract ran at >500 kDa, after incubation with the mucinase cocktail there was a reduction in high molecular weight species and the appearance of a new band at ~200 kDa. When the extract was incubated with L. pneumophila 130b wild-type supernatant there was again a reduction of high molecular weight material but with the addition of a new fragment at ~95 kDa. On the other hand, the chiA mutant supernatant produced a profile that was more similar to the control, although there was evidence of another new species at ~100 kDa. When the experiment was performed using a greater amount of mucin, the difference between the wild-type and mutant was even more evident (Fig 5A, right panel). Three clear fragments (~100 kDa, ~95 kDa and ~90 kDa) could be observed in the type II mucin extract incubated with wild-type supernatants, with the middle band absent when incubated with the chiA mutant supernatant. This band is dependent upon ChiA and implies that ChiA can function as both chitinase and a mucinase. Furthermore, the presence of the ~100 kDa and ~90 kDa fragments in mucin extracts treated with either wild-type or chiA mutant supernatants suggests that L. pneumophila secretes additional mucinase enzymes that are yet to be identified. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. Mucinase activity of ChiA. (A) Secreted mucinase activity of L. pneumophila wild-type and chiA mutant strains. Left panel: immunoblot of type II porcine stomach mucins (200 μg) incubated with either BYE medium alone (BYE), a cocktail of known mucinase enzymes added to BYE medium (cocktail), or supernatants from BYE cultures of wild-type 130b (WT) or chiA mutant NU318 (ΔchiA). Asterisk highlights a lower-MW (~200 kDa) mucin species generated by the cocktail that is not present in the supernatant samples. Right panel: immunoblot of type II porcine stomach mucins (400 μg) incubated with either supernatants from BYE cultures of wild-type 130b (WT) or chiA mutant NU318 (ΔchiA). White arrow highlights ChiA-dependent mucin fragment (~95 kDa) and black arrows highlight non-ChiA-dependent mucin fragments (~100 and ~90 kDa). The data presented are representative of three independent experiments. (B) Mucin penetration assay of L. pneumophila wild-type (WT) and chiA mutant (ΔchiA) strains applied to the upper chamber of 3.0 μm transwell coated with type II mucin extract. Bacteria that penetrated the transwell were collected from the lower chamber and plated for CFU. Penetration ratio represents CFU in lower chamber 50 or 100 μg mucin / CFU in lower chamber 0 μg mucin. N = 3 experimental replicates. Statistical analysis was done using Two-way ANOVA with Boneferri post-hoc test. Error bars represent standard deviation. *P = <0.05, **P = <0.01. (C) Immunoblot of type II porcine stomach mucin extract incubated with ChiA-FL, subdomains (NTD, CTD), SslE or buffer alone, +/- EDTA, detected with MUC5AC antibody. The bands at ~70 kDa and ~60 kDa correspond to ChiA and SslE processed MUC5AC fragments, respectively. https://doi.org/10.1371/journal.ppat.1008342.g005 Mucins are high molecular weight glycoproteins that contain large numbers of heavily O-glycosylated serine/threonine rich repeat sequences [42]. They exist as cell surface exposed transmembrane proteins or secreted gel-forming proteins of the mucosal barrier and act as a first line of defence against bacterial infection [43]. The normal stomach mucosa is characterised by expression of MUC1, MUC5AC, and MUC6 mucins [44], however, MUC1 and MUC5AC are also major mucins expressed in the mammalian airway and lung [45]. Therefore, to determine whether ChiA can facilitate mucin penetration of L. pneumophila we performed an artificial mucin penetration assay. After 2 hours incubation with either L. pneumophila wild-type 130b or chiA mutant NU318, we observed a 2.7- and 2.4-fold decrease in the number of colonies from the chiA mutant compared with wild-type in the presence of 50 and 100 μg type II mucin extract, respectively (Fig 5B). This confirms that ChiA promotes L. pneumophila dissemination through mucin layers. We then assessed whether the degradation of mucins by ChiA could also be a source of nutrients for L. pneumophila. However, L. pneumophila chiA mutant NU318 grown in chemically defined media, supplemented with up to 100 μg of type II mucin extract, displayed identical growth patterns to the wild-type strain (S8 Fig). This indicates that ChiA-dependent degradation of mucins is not important for L. pneumophila growth. ChiA-CTD is a Zn2+-dependent peptidase We then tested the ability of recombinant ChiA to specifically degrade MUC5AC within type II mucin extracts by immunoblotting and compared its profile to that of recombinant E. coli SslE. Intact MUC5AC did not enter the gel in the buffer controls and this was likely due to its high carbohydrate content and large mass (>500 kDa before glycosylation). However, incubation of type II mucin extract with ChiA-FL and ChiA-CTD, but not ChiA-NTD, resulted in the processing of MUC5AC into a new ~70 kDa fragment (Fig 5C). When the mucin extract was incubated with SslE, MUC5AC was processed into a different ~60 kDa species. SslE is a member of the M60 family of metalloproteases [38], which use a HExxH motif to coordinates Zn2+ in their active site [46]. In the presence of the metal chelating agent ethylenediaminetetraacetic acid (EDTA) we did not detect activity for SslE or ChiA-CTD and this clearly shows that the C-terminal domain of ChiA has dual enzymatic activity. This also suggests that ChiA and SslE use a similar peptidase mechanism for the degradation of mucins. To evaluate this further we performed Molecular Dynamics (MD) simulations and examined the ability of ChiA-CTD to bind Zn2+ in silico. The protein was ‘soaked’ in a water solution at high Zn2+ concentration and the system was then left to evolve over time to identify the regions on the protein surface where Zn2+ ions tend to bind. Multiple short simulations were run starting from different random placements of Zn2+ ions, for an aggregated simulation time of 1.7 μs. Analysis of the Zn2+ spatial distribution function (sdf) calculated on the concatenated trajectories highlighted multiple high Zn2+ density sites in the region around the chitinase active site, providing information on the different ways in which Zn2+ could bind to the protein in this region. The highest density was found at the chitinase active site (region 1), where Zn2+ is coordinated by Asp541, Glu543 and Gln617, with two other sites in close proximity (regions 2,3) coordinated by Glu543 and Gln583 (Fig 6). Binding of two Zn2+ions in the active site of Bacillus cereus ChiNCTU2 has been shown to inhibit chitinase activity [27] and indicates that metal binding could modulate the different enzyme activities in ChiA. A unique cluster of Zn2+ sites was also located away from the chitinase active site, near the MPD-2 ligand site in the ChiA-CTD crystal structure, involving residues Asp504 (region 4), His544 (region 5), Glu543 and Gln595 (region 6), Asn547 (region 7) and His506 (region 8) (Fig 6). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 6. ChiA-CTD Zn2+ binding sites. Surface representation of ChiA-CTD showing the spatial distribution of Zn2+ ions during MD simulations. The sdf is represented with isosurfaces connecting points with sdf = 20 (green mesh), 25 (yellow mesh) and 30 (red surface) x average sdf. Zn2+ high-density sites (red surface) around the chitinase and peptidase active sites are numbered 1 to 8. Blow out boxes show representative structures from the MD simulations to illustrate Zn2+ binding in the eight regions, with Zn2+ ions shown as spheres, their coordinating residues as sticks and ChiA-CTD as cartoon. https://doi.org/10.1371/journal.ppat.1008342.g006 To verify these in silico observation we used isothermal titration calorimetry (ITC) and measured an equilibrium dissociation constant (KD) of 556 nM for approximately three Zn2+ ions (N = 3.04) binding to wild-type ChiA-CTD (Fig 7). During the experiment we observed both exothermic and endothermic signals, which were not detected in reverse or blank titrations (S9 Fig). This may reflect a different binding mechanism for each site. We then examined binding of Zn2+ to ChiA-CTDE543M and observed exothermic binding with a ~1.6-fold reduction in affinity (KD 890 nM) at a single site (N = 1.07). This indicated that Glu543 is involved in the coordination of zinc in the chitinase active site. To assess whether residues from the second cluster also formed a genuine binding site, we created E543M/D504A, E543M/H506A, E543M/H544A, E543M/N547A and E543M/Q595A double mutants in ChiA-CTD (S2 Fig). When we assessed Zn2+ binding to ChiA-CTDE543M/D504A using ITC we measured exothermic binding with a further ~1.5-fold reduction in affinity (KD 1.3 μM) at a single site (N = 1.28). However, examination of Zn2+ binding to ChiA-CTDE543M/H506A, ChiA-CTDE543M/H544A, ChiA-CTDE543M/N547A and ChiA-CTDE543M/DQ595A showed no binding. This indicates that His506, His544, Asn547 and Q595 form a unique zinc binding site in ChiA. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 7. ITC analysis of the interaction of Zn2+ with ChiA-CTD. Isothermal titration calorimetry was used to measure the affinities of Zn2+ for wild-type ChiA-CTD (WT) and ChiA-CTD variants (E543M, E543M/D504A, E543M/H506A, E543M/H544A, E543M/N547A and E543M/Q595A). Raw data (top) and normalized binding curves (bottom) are reported after subtraction of blank heat pulses. Black squares indicate the normalized heat of interaction obtained per injection, while a black curve represents the best fit obtained by non-linear least-squares procedures based on a 1:1 binding model. https://doi.org/10.1371/journal.ppat.1008342.g007 ChiA-CTD uses a novel mechanism to cleave mucin-like glycoproteins To assess the role of these residues in the degradation of MUC5AC, we created D504A, H506A, H544A, N547A and Q595A single site mutations in ChiA-CTD. All constructs were well folded, retained their ability to bind chitin in pull-down experiments and with our existing single site ChiA variants (ChiA-CTDE543M, ChiA-CTDQ583A and CTDQ617A), they were still able to bind immobilized type II and III mucin extracts in ELISA assays (S2, S10 and S11 Figs). We then incubated these proteins with type II mucin extract and inspected their MUC5AC degradation profiles by immunoblotting (Fig 8A). Incubation with ChiA-CTD and ChiA-CTDE543M both produced identical MUC5AC degradation patterns, while all other ChiA-CTD variants showed no activity. Human C1-inhibitor (C1-INH) is a serine protease inhibitor with a major role in regulating the contact activation pathway, the classical complement pathway and the lectin complement pathway [47]. C1-INH contains mucin-like glycosylation patterns [48] and is cleaved by other mucin metalloproteases such as enterohemorrhagic E. coli StcE [49]. When we incubated C1-INH with ChiA-CTD or ChiA-CTDE543M we again measured peptidase activity with a new C1-INH fragment appearing at ~30 kDa. Likewise, in the presence of the other ChiA-CTD mutants we detected no activity (Fig 8B). This demonstrates that ChiA-CTD has broad specificity for mucin-like glycoproteins and its peptidase activity is independent from the adjacent chitinase active site. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 8. Peptidase active site of ChiA. (A) Immunoblot of type II porcine stomach mucin extract incubated with ChiA-CTD, ChiA-CTD mutants (D504A, H506A, E543M, H544A, N547A, Q583A, Q595A, 617A) or buffer alone and detected with MUC5AC antibody. The band at ~75 kDa corresponds to a ChiA processed MUC5AC fragment. (B) Immunoblot of human C1-INH incubated with ChiA-CTD, ChiA-CTD mutants (D504A, H506A, E543M, H544A, N547A, Q583A, Q595A, 617A) or buffer alone and detected with Pro-Q Emerald 300 glycoprotein stain. The band at ~30 kDa corresponds to a ChiA processed C1-INH fragment. (C) Surface and cartoon representation of ChiA-CTD bound to MPD-2 (cyan; spheres and sticks) and modelled chitotetraose (yellow; spheres and sticks), highlighting the potential for mucin branched glycan recognition. Residues that bind Zn2+ or form the metal-dependent aminopeptidase active site are highlighted (purple: chitinase function; red: peptidase Zn2+ binding; blue: peptidase general base). https://doi.org/10.1371/journal.ppat.1008342.g008 ChiA is a multi-domain protein Full-length ChiA from L. pneumophila 130b (ChiA-FL; numbered 1–762 for the mature protein; NCBI accession WP_072401826.1) with an N-terminal His6-tag was produced in Escherichia coli K12 and purified by nickel-affinity and size exclusion chromatography. Despite extensive screening ChiA-FL resisted crystallization and we therefore used bioinformatics analysis to produce a series of subdomain constructs for further characterization (Fig 1A). While previous examination of the C-terminal domain of ChiA (ChiA-CTD: residues 419–762) had revealed high primary sequence homology to other GH18 chitinase domains [17], the N-terminal region (ChiA-NT; residues 1–417) contains unique primary sequence with no significant homology to any other known protein. Nonetheless, through secondary structure prediction [22] and template based modelling using the Phyre2 [23] and Robetta [24] servers, we identified three putative N-terminal subdomains based on predicted structural similarity with carbohydrate-binding modules (CBMs; ChiA-N1: residues 1–140), fibronectin type-III-like domains (Fn3; ChiA-N2: residues 138–299) and a chitinase A N-terminal domain (ChiN; ChiA-N3: residues 285–417) (Fig 1A and S1 Table). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Chitin binding and endochitinase functions of ChiA. (A) Schematic representation of ChiA with domain boundaries annotated. (B) ChiA-FL, subdomains (NT, N1, N2, N3, CTD) and ChiA-CTD mutants (E543M, Q583A, Q617A) were assayed for chitinase activity against p-NP-[GlcNAc]3. Data represent the mean and standard deviation for triplicate experiments. (C) Chitin pull-down experiment to assess direct interactions between immobilized chitin and ChiA. ChiA-FL and subdomains (NT, N1, N2, N3, CTD, CTDE543M) were incubated with chitin beads and analysed by SDS-PAGE. BSA was used as a control. L: loaded sample; B: eluted beads. Eluted samples undergo an upward shift compared to the input sample due to differences in buffer conditions. Data is representative of three independent repeat experiments. https://doi.org/10.1371/journal.ppat.1008342.g001 To examine the function of the ChiA N-terminal domains we began by examining their endochitinase activity [17]. Each construct was expressed with an N-terminal His6-tag in E. coli K12 and purified by nickel-affinity and size exclusion chromatography. All reagents were well folded as determined by 1D 1H nuclear magnetic resonance (NMR) spectroscopy (S1 Fig). As expected, ChiA-FL and ChiA-CTD were both active against p-nitrophenyl β-D-N,N′,N″ triacetylchitotriose (pNP-[GlcNAc]3) but no activity was detected for ChiA-NT or an E543M ChiA-CTD active site mutant (ChiA-CTDE543M) (Fig 1B and S2 Fig). We then assayed binding of ChiA sub-domains to immobilized chitin and observed that in addition to ChiA-CTD, the N1-domain also recognizes chitin polymers, which supports its role as a carbohydrate binding module (Fig 1C). Atomic structure of ChiA-CTD We next initiated crystallographic studies of the ChiA subdomains. We readily obtained crystals for ChiA-CTD and its structure was determined using iodide single isomorphous replacement with anomalous scattering (I-SIRAS) phasing. Electron-density maps were refined to 1.7 Å (Table 1) and the final model contains two identical chains, with all molecules built except for the N-terminal His6-tags and adjacent ChiA-CTD residues Val419 to Gly424. Each chain forms an anticipated GH18 α/β-fold and is composed of 11 β-strands and 13 α-helices (Fig 2A). High concentrations of 2-methyl-2,4-pentanediol (MPD) were used as a precipitant during crystallization and we observed four MPD molecules in the final model; one bound to the catalytic Asp541 and Glu543 residues (MPD-1) and one within a hydrophobic pocket formed by the α5 helix and α5-α5′/β5-β6′ loops (MPD-2) (S3 Fig). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Crystal structure of ChiA-CTD. (A) Cartoon representation of ChiA-CTD with secondary structure and extended loops annotated. Additional ChiA-CTD α3-helix is highlighted with a red asterisk. (B) Stick representation of the ChiA-CTD active site (green) superimposed with ChiNCTU2 E145G/Y227F mutant (grey) in complex with chitotetraose (yellow) (PDB ID code 3n18) [27]. Mutated residues in ChiNCTU2 are indicated and carbohydrate positions relative to the hydrolysed glycosidic bond are numbered. (C) Model of ChiA-CTD shown as cartoon and (D) electrostatic surface potential, bound to chitotetraose drawn as spheres. https://doi.org/10.1371/journal.ppat.1008342.g002 Download: PPT PowerPoint slide PNG larger image TIFF original image Table 1. Diffraction data and refinement statistics. https://doi.org/10.1371/journal.ppat.1008342.t001 The overall structure of ChiA-CTD is highly similar to other GH18 chitinase domains, and the Dali server [26] identified Bacillus cereus ChiNCTU2 enzyme inactive E145G/Y227F mutant in complex with chitotetraose (Protein Data Bank (PDB) ID code 3n18) [27]; Bacillus anthracis Chi36 (PDB ID code 5kz6); Chromobacterium violaceum ChiA (PDB ID code 4tx8); and Streptomyces coelicolor ChiA (PDB ID code 3ebv) as having the highest homologies (Z score: 36.3, 35.9, 34.4, 34.1 respectively; rmsd: 2.2 Å, 2.3 Å, 2.2 Å, 1.8 Å, respectively). The chitinase active sites of ChiNCTU2 and ChiA-CTD have high primary sequence identity and tertiary structure homology (Fig 2B and S4 Fig) and modelling of chitotetraose binding indicates that chitin lines a negatively charged valley on the surface of ChiA-CTD (Fig 2C and 2D). Chitotetraose overlays with MPD-1 in the active site (S3 Fig) and the MPD-2 site is positioned adjacent to the reducing end of the modelled chitotetraose. In this binding model, Gln583 and Gln617 have a central role in the correct positioning of chitotetraose in the ChiA-CTD active site. We therefore created Q583A and Q617A mutants in recombinant ChiA-CTD and as anticipated, these mutants showed no activity against pNP-[GlcNAc]3 (Fig 1B and S2 Fig). However, L. pneumophila ChiA-CTD also possesses unique features that are not observed in homologous structures. These include an additional α-helix (α3), an extended β3-α3 loop, an extended α6-α6′ loop and an extended β7-α7 loop (Fig 2A and S4 and S5 Figs). ChiA is an elongated and dynamic structure in solution We used small angle X-ray scattering (SAXS) to model the global structure of full-length ChiA in solution. Four different concentrations at 4, 2, 1, and 0.5 mg/ml were measured but signs of aggregation were apparent at concentrations above 1 mg/ml (S6 Fig). To achieve the highest signal/noise ratio, all further analysis was carried out with the data from the 1 mg/ml sample (S6 Fig and S2 Table). Guinier analysis suggested a radius of gyration (Rg), the root mean square distance to the particles centre of mass, of 5.43 nm and analysis of the distance distribution function (P(r)) suggested a maximum particle dimension (Dmax) of 17.77 nm and Rg of 5.45 nm (S6 Fig). Using BSA as a standard, we calculated a particle molecular mass of 89.2 kDa, which is within the method error range for a monomeric 82.6 kDa ChiA. Kratky, Kratky-Debye and Porod-Debye plot analyses of the SAXS data indicated that ChiA is a highly dynamic particle in solution (S6 Fig) [28]. This is likely due to flexibility within the ChiA inter-domain linkers and we therefore used the ensemble optimization method (EOM) to determine molecular model ensembles of ChiA that best fit the SAXS data [29]. As we were not able to obtain crystals for ChiA N-domains, an initial model of ChiA was created using a Phyre2 derived N1-domain (residues 22–147), a Robetta derived N2-domain (encompassing two further subdomains: residues 152–245 and 248–305), a Phyre2 derived ChiA N3-domain (residues 315–414) and the crystal structure of ChiA-CTD (residues 439–777), separated by flexible linkers (Fig 3A and S1 Table) [23, 24, 29]. Ensemble optimization analysis of the scattering data yielded an excellent fit between experimental and calculated SAXS profiles (χ2: 1.1), which again indicates that ChiA is highly flexible in solution (Rflex 91.4) [30] with conformation ensembles clustered within three populations (Fig 3B and 3C and S3 Table). The majority of the simulated conformations exhibited partially extended or fully extended structures at Rg 44–56 Å or Rg 60–71 Å, respectively, whereas minor conformations of closed structures were also populated at Rg 33–43 Å. This analysis provides experiential support for the global features of our ChiA N-domain models but also suggests that there is cooperation between ChiA domains. As inter-domain flexibility is a key feature of processive enzymes [31], these data suggest that processivity may also be important for the function of ChiA. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. Model of ChiA-FL in solution. (A) Initial model of ChiA generated by EOM 2.0 [30]. Linkers are shown as grey spheres. (B) EOM fit (orange line) to the ChiA SAXS data (black open circles) with χ2 of 1.09. (C) Three independent ensemble optimization method runs (yellow, orange and burgundy) yielded similar distributions of three populations. Sample ChiA models corresponding to the centre of each population for all three runs are shown. https://doi.org/10.1371/journal.ppat.1008342.g003 ChiA is targeted to the L. pneumophila surface Although we have repeatedly detected secreted ChiA in bacterial culture supernatants [17, 32], it has been well documented in other bacteria that some type II substrates associate with the bacterial surface upon their secretion [11, 33]. To determine whether ChiA is also targeted to the bacterial surface, L. pneumophila 130b was examined by whole-cell ELISA using anti-ChiA, anti-Mip and anti-ProA antibodies [32, 34] (Fig 4A). Both ChiA and Mip, a known surface exposed protein [35], were positive by ELISA, whilst the metalloprotease ProA, another T2SS substrate [10, 36], was negative. As the translocation signal for ChiA (and T2SS substrates in general) has yet to be determined [10, 11], in vivo truncations or site directed mutagenesis of chiA could inadvertently impede the secretion of ChiA. Therefore, to examine which region of ChiA is responsible for this localization, a L. pneumophila NU318 (chiA) mutant [17] was incubated with recombinant ChiA fragments and the whole-cell ELISA was repeated using ChiA antisera (Fig 4B). ChiA-NT and the N3-domain exhibited significant binding to the bacterial surface while no binding was observed for ChiA-CTD, ChiA-N1 or ChiA-N2. A direct comparison between anti-ChiA antibodies binding to ChiA-FL, ChiA-NT and ChiA-CTD indicated that the antiserum binds to the N- and C-terminal fragments at a similar or higher level than to full length ChiA (S7 Fig). Likewise, direct comparison between the three N-terminal subdomains revealed that anti-ChiA antibodies bind to the N1 and N2 subdomains with comparable or higher affinity as they do to ChiA-N3 (S7 Fig). Thus, the greater association of ChiA-FL, ChiA-NT and ChiA-N3 with the L. pneumophila surface, as detected by whole-cell ELISA (Fig 4B), is not simply the result of a reduced ability of the antiserum to recognize ChiA-CTD, ChiA-N1, and ChiA-N2. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 4. L. pneumophila surface association of ChiA and mucin binding. (A) Whole cell ELISA of L. pneumophila wild-type 130b (WT), chiA mutant NU318 (chiA), proA mutant AA200 (proA) and mip mutant NU203 (mip) detected with antiserum specific for either ChiA, ProA, or Mip. (B) Whole cell ELISA of chiA mutant incubated with either recombinant ChiA-FL or subdomains (NT, N1, N2, N3, CTD) and detected with antiserum specific for ChiA. PBS buffer alone was used as a control (-). Multiple comparisons against the control were made using a one-way ANOVA, Holm-Šídák multiple comparisons test; * P < 0.05, *** P < 0.001. (C) ELISA analysis of binding between immobilised type I-S, II or III porcine stomach mucins and His-tagged ChiA-FL, subdomains (NT, N1, N2, N3, CTD) and controls (SslE, NttE) detected with anti-His-tag antibody. BSA-coated wells were used as controls. *** P < 0.001; verses control empty well by two-tailed Student’s test. (D) Mucin binding to GFP-expressing L. pneumophila WT or chiA mutant strains were incubated at 25°C or 37°C with PBS, type I-S, II or III mucins followed by Texas Red-tagged wheat germ agglutinin (WGA). Mucin binding to bacteria was quantified by flow cytometry. * P < 0.05; verses PBS control by two-tailed Student’s test. All data represent the mean and standard deviation for triplicate experiments. https://doi.org/10.1371/journal.ppat.1008342.g004 ChiA is a mucin binding protein Some bacterial chitinases and chitin binding proteins are able to promote infection through adhesion to and/or degradation of host glycoconjugates [37] and we hypothesized that ChiA may interact with exogenous mucins in the lungs and elsewhere. We therefore examined the binding capacity of recombinant ChiA-FL, ChiA domains, L. pneumophila NttE, another T2SS substrate [10, 17], and E. coli SslE, a known mucin binding protein and mucinase [38], to immobilized commercially available mucin extracts by ELISA using anti-His antibodies (Fig 4C). All ChiA samples and SslE displayed significant adhesion to mucins isolated from porcine stomachs (type II and III), but this was not observed with a mucin extract from bovine submaxillary glands (type I-S). Conversely, NttE showed no binding to any of the mucin samples. This confirmed that ChiA has additional specificity for non-chitinous ligands and implied that ChiA could bind host glycoproteins on the Legionella surface. To assess this, we incubated L. pneumophila 130b wild-type and NU318 (chiA) mutant strains with type I-S, II and III mucin extracts followed by wheat germ agglutinin and measured their binding to the bacterial surface by flow cytometry. Whether examined at 25°C or 37°C, type II and III extracts showed strong association to both L. pneumophila strains, but submaxillary gland mucins did not (Fig 4D). However, there was no significant difference in binding of the type II and III mucins between wild-type and chiA mutant strains, which indicates that other factors apart from ChiA are present on the Legionella surface that can also recognize mucins and in the case of the mutant, compensate for the loss of ChiA. Interestingly, the mean binding of L. pneumophila to both type II and III mucins trends slightly higher in the chiA mutant strain and we speculated that this may be due to ChiA degrading mucins on the bacterial surface on the wild-type strain. ChiA increases penetration of L. pneumophila through the mucin layer We next examined whether secreted ChiA is able to degrade mucins. Porcine stomach type II mucin extract was incubated with supernatants from L. pneumophila 130b wild-type and NU318 (chiA) mutant strains or a cocktail of enzymes (pepsin, pronase, β-N-acetylglucosaminidase, fucosidase) with known activity against mucins [39–41], and then analysed by immunoblotting using wheat germ agglutinin (Fig 5A, left panel). While the majority of the mucin extract ran at >500 kDa, after incubation with the mucinase cocktail there was a reduction in high molecular weight species and the appearance of a new band at ~200 kDa. When the extract was incubated with L. pneumophila 130b wild-type supernatant there was again a reduction of high molecular weight material but with the addition of a new fragment at ~95 kDa. On the other hand, the chiA mutant supernatant produced a profile that was more similar to the control, although there was evidence of another new species at ~100 kDa. When the experiment was performed using a greater amount of mucin, the difference between the wild-type and mutant was even more evident (Fig 5A, right panel). Three clear fragments (~100 kDa, ~95 kDa and ~90 kDa) could be observed in the type II mucin extract incubated with wild-type supernatants, with the middle band absent when incubated with the chiA mutant supernatant. This band is dependent upon ChiA and implies that ChiA can function as both chitinase and a mucinase. Furthermore, the presence of the ~100 kDa and ~90 kDa fragments in mucin extracts treated with either wild-type or chiA mutant supernatants suggests that L. pneumophila secretes additional mucinase enzymes that are yet to be identified. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. Mucinase activity of ChiA. (A) Secreted mucinase activity of L. pneumophila wild-type and chiA mutant strains. Left panel: immunoblot of type II porcine stomach mucins (200 μg) incubated with either BYE medium alone (BYE), a cocktail of known mucinase enzymes added to BYE medium (cocktail), or supernatants from BYE cultures of wild-type 130b (WT) or chiA mutant NU318 (ΔchiA). Asterisk highlights a lower-MW (~200 kDa) mucin species generated by the cocktail that is not present in the supernatant samples. Right panel: immunoblot of type II porcine stomach mucins (400 μg) incubated with either supernatants from BYE cultures of wild-type 130b (WT) or chiA mutant NU318 (ΔchiA). White arrow highlights ChiA-dependent mucin fragment (~95 kDa) and black arrows highlight non-ChiA-dependent mucin fragments (~100 and ~90 kDa). The data presented are representative of three independent experiments. (B) Mucin penetration assay of L. pneumophila wild-type (WT) and chiA mutant (ΔchiA) strains applied to the upper chamber of 3.0 μm transwell coated with type II mucin extract. Bacteria that penetrated the transwell were collected from the lower chamber and plated for CFU. Penetration ratio represents CFU in lower chamber 50 or 100 μg mucin / CFU in lower chamber 0 μg mucin. N = 3 experimental replicates. Statistical analysis was done using Two-way ANOVA with Boneferri post-hoc test. Error bars represent standard deviation. *P = <0.05, **P = <0.01. (C) Immunoblot of type II porcine stomach mucin extract incubated with ChiA-FL, subdomains (NTD, CTD), SslE or buffer alone, +/- EDTA, detected with MUC5AC antibody. The bands at ~70 kDa and ~60 kDa correspond to ChiA and SslE processed MUC5AC fragments, respectively. https://doi.org/10.1371/journal.ppat.1008342.g005 Mucins are high molecular weight glycoproteins that contain large numbers of heavily O-glycosylated serine/threonine rich repeat sequences [42]. They exist as cell surface exposed transmembrane proteins or secreted gel-forming proteins of the mucosal barrier and act as a first line of defence against bacterial infection [43]. The normal stomach mucosa is characterised by expression of MUC1, MUC5AC, and MUC6 mucins [44], however, MUC1 and MUC5AC are also major mucins expressed in the mammalian airway and lung [45]. Therefore, to determine whether ChiA can facilitate mucin penetration of L. pneumophila we performed an artificial mucin penetration assay. After 2 hours incubation with either L. pneumophila wild-type 130b or chiA mutant NU318, we observed a 2.7- and 2.4-fold decrease in the number of colonies from the chiA mutant compared with wild-type in the presence of 50 and 100 μg type II mucin extract, respectively (Fig 5B). This confirms that ChiA promotes L. pneumophila dissemination through mucin layers. We then assessed whether the degradation of mucins by ChiA could also be a source of nutrients for L. pneumophila. However, L. pneumophila chiA mutant NU318 grown in chemically defined media, supplemented with up to 100 μg of type II mucin extract, displayed identical growth patterns to the wild-type strain (S8 Fig). This indicates that ChiA-dependent degradation of mucins is not important for L. pneumophila growth. ChiA-CTD is a Zn2+-dependent peptidase We then tested the ability of recombinant ChiA to specifically degrade MUC5AC within type II mucin extracts by immunoblotting and compared its profile to that of recombinant E. coli SslE. Intact MUC5AC did not enter the gel in the buffer controls and this was likely due to its high carbohydrate content and large mass (>500 kDa before glycosylation). However, incubation of type II mucin extract with ChiA-FL and ChiA-CTD, but not ChiA-NTD, resulted in the processing of MUC5AC into a new ~70 kDa fragment (Fig 5C). When the mucin extract was incubated with SslE, MUC5AC was processed into a different ~60 kDa species. SslE is a member of the M60 family of metalloproteases [38], which use a HExxH motif to coordinates Zn2+ in their active site [46]. In the presence of the metal chelating agent ethylenediaminetetraacetic acid (EDTA) we did not detect activity for SslE or ChiA-CTD and this clearly shows that the C-terminal domain of ChiA has dual enzymatic activity. This also suggests that ChiA and SslE use a similar peptidase mechanism for the degradation of mucins. To evaluate this further we performed Molecular Dynamics (MD) simulations and examined the ability of ChiA-CTD to bind Zn2+ in silico. The protein was ‘soaked’ in a water solution at high Zn2+ concentration and the system was then left to evolve over time to identify the regions on the protein surface where Zn2+ ions tend to bind. Multiple short simulations were run starting from different random placements of Zn2+ ions, for an aggregated simulation time of 1.7 μs. Analysis of the Zn2+ spatial distribution function (sdf) calculated on the concatenated trajectories highlighted multiple high Zn2+ density sites in the region around the chitinase active site, providing information on the different ways in which Zn2+ could bind to the protein in this region. The highest density was found at the chitinase active site (region 1), where Zn2+ is coordinated by Asp541, Glu543 and Gln617, with two other sites in close proximity (regions 2,3) coordinated by Glu543 and Gln583 (Fig 6). Binding of two Zn2+ions in the active site of Bacillus cereus ChiNCTU2 has been shown to inhibit chitinase activity [27] and indicates that metal binding could modulate the different enzyme activities in ChiA. A unique cluster of Zn2+ sites was also located away from the chitinase active site, near the MPD-2 ligand site in the ChiA-CTD crystal structure, involving residues Asp504 (region 4), His544 (region 5), Glu543 and Gln595 (region 6), Asn547 (region 7) and His506 (region 8) (Fig 6). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 6. ChiA-CTD Zn2+ binding sites. Surface representation of ChiA-CTD showing the spatial distribution of Zn2+ ions during MD simulations. The sdf is represented with isosurfaces connecting points with sdf = 20 (green mesh), 25 (yellow mesh) and 30 (red surface) x average sdf. Zn2+ high-density sites (red surface) around the chitinase and peptidase active sites are numbered 1 to 8. Blow out boxes show representative structures from the MD simulations to illustrate Zn2+ binding in the eight regions, with Zn2+ ions shown as spheres, their coordinating residues as sticks and ChiA-CTD as cartoon. https://doi.org/10.1371/journal.ppat.1008342.g006 To verify these in silico observation we used isothermal titration calorimetry (ITC) and measured an equilibrium dissociation constant (KD) of 556 nM for approximately three Zn2+ ions (N = 3.04) binding to wild-type ChiA-CTD (Fig 7). During the experiment we observed both exothermic and endothermic signals, which were not detected in reverse or blank titrations (S9 Fig). This may reflect a different binding mechanism for each site. We then examined binding of Zn2+ to ChiA-CTDE543M and observed exothermic binding with a ~1.6-fold reduction in affinity (KD 890 nM) at a single site (N = 1.07). This indicated that Glu543 is involved in the coordination of zinc in the chitinase active site. To assess whether residues from the second cluster also formed a genuine binding site, we created E543M/D504A, E543M/H506A, E543M/H544A, E543M/N547A and E543M/Q595A double mutants in ChiA-CTD (S2 Fig). When we assessed Zn2+ binding to ChiA-CTDE543M/D504A using ITC we measured exothermic binding with a further ~1.5-fold reduction in affinity (KD 1.3 μM) at a single site (N = 1.28). However, examination of Zn2+ binding to ChiA-CTDE543M/H506A, ChiA-CTDE543M/H544A, ChiA-CTDE543M/N547A and ChiA-CTDE543M/DQ595A showed no binding. This indicates that His506, His544, Asn547 and Q595 form a unique zinc binding site in ChiA. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 7. ITC analysis of the interaction of Zn2+ with ChiA-CTD. Isothermal titration calorimetry was used to measure the affinities of Zn2+ for wild-type ChiA-CTD (WT) and ChiA-CTD variants (E543M, E543M/D504A, E543M/H506A, E543M/H544A, E543M/N547A and E543M/Q595A). Raw data (top) and normalized binding curves (bottom) are reported after subtraction of blank heat pulses. Black squares indicate the normalized heat of interaction obtained per injection, while a black curve represents the best fit obtained by non-linear least-squares procedures based on a 1:1 binding model. https://doi.org/10.1371/journal.ppat.1008342.g007 ChiA-CTD uses a novel mechanism to cleave mucin-like glycoproteins To assess the role of these residues in the degradation of MUC5AC, we created D504A, H506A, H544A, N547A and Q595A single site mutations in ChiA-CTD. All constructs were well folded, retained their ability to bind chitin in pull-down experiments and with our existing single site ChiA variants (ChiA-CTDE543M, ChiA-CTDQ583A and CTDQ617A), they were still able to bind immobilized type II and III mucin extracts in ELISA assays (S2, S10 and S11 Figs). We then incubated these proteins with type II mucin extract and inspected their MUC5AC degradation profiles by immunoblotting (Fig 8A). Incubation with ChiA-CTD and ChiA-CTDE543M both produced identical MUC5AC degradation patterns, while all other ChiA-CTD variants showed no activity. Human C1-inhibitor (C1-INH) is a serine protease inhibitor with a major role in regulating the contact activation pathway, the classical complement pathway and the lectin complement pathway [47]. C1-INH contains mucin-like glycosylation patterns [48] and is cleaved by other mucin metalloproteases such as enterohemorrhagic E. coli StcE [49]. When we incubated C1-INH with ChiA-CTD or ChiA-CTDE543M we again measured peptidase activity with a new C1-INH fragment appearing at ~30 kDa. Likewise, in the presence of the other ChiA-CTD mutants we detected no activity (Fig 8B). This demonstrates that ChiA-CTD has broad specificity for mucin-like glycoproteins and its peptidase activity is independent from the adjacent chitinase active site. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 8. Peptidase active site of ChiA. (A) Immunoblot of type II porcine stomach mucin extract incubated with ChiA-CTD, ChiA-CTD mutants (D504A, H506A, E543M, H544A, N547A, Q583A, Q595A, 617A) or buffer alone and detected with MUC5AC antibody. The band at ~75 kDa corresponds to a ChiA processed MUC5AC fragment. (B) Immunoblot of human C1-INH incubated with ChiA-CTD, ChiA-CTD mutants (D504A, H506A, E543M, H544A, N547A, Q583A, Q595A, 617A) or buffer alone and detected with Pro-Q Emerald 300 glycoprotein stain. The band at ~30 kDa corresponds to a ChiA processed C1-INH fragment. (C) Surface and cartoon representation of ChiA-CTD bound to MPD-2 (cyan; spheres and sticks) and modelled chitotetraose (yellow; spheres and sticks), highlighting the potential for mucin branched glycan recognition. Residues that bind Zn2+ or form the metal-dependent aminopeptidase active site are highlighted (purple: chitinase function; red: peptidase Zn2+ binding; blue: peptidase general base). https://doi.org/10.1371/journal.ppat.1008342.g008 Discussion Chitin is highly abundant in the environment and can function as a source of carbon and nitrogen [50] but several chitinases have been identified as key virulence factors in bacterial disease [37]. These include Enterococcus faecalis efChiA, E. coli ChiA, Vibrio cholerae ChiA2, Francisella tularensis ChiA, Listeria monocytogenes ChiA and ChiB, Pseudomonas aeruginosa ChiC, Salmonella typhimurium ChiA and L. pneumophila ChiA. Although it is unclear how these enzymes perform these dual functions, there is strong evidence that they interact with host glycoconjugates and through their localization and/or enzymatic activity are able to modulate host defence mechanisms [37]. We have determined that L. pneumophila ChiA has activity against human C1-INH and porcine stomach derived mucins, and the degradation of MUC5AC produces a similar degradation profile to the M60-family E. coli zinc-aminopeptidase SslE [51, 52]. Recent structural analysis of Bacteroides thetaiotaomicron BT4244, Pseudomonas aeruginosa IMPa and Clostridium perfringens ZmpB M60 proteins has revealed unique structural adaptations that allow them to accommodate different glycan sequences while all cleaving the peptide bond immediately preceding the glycosylated residue [46]. Similarly, E. coli StcE is an M66-family zinc metalloprotease that recognizes distinct peptide and glycan motifs in mucin-like proteins and then cleaves the peptide backbone using an extended HExxHxxGxxH motif [53, 54]. In StcE, three histidine residues in the conserved motif act as ligands for a single catalytic zinc, while in M60 enzymes two histidines and another residue perform this role [46, 53]. A nucleophilic water molecule is the fourth ligand for the zinc, and this is coordinated by a conserved glutamate, which acts as a general base during catalysis. We have shown that L. pneumophila ChiA functions in a similar fashion to SslE and StcE but as it does not contain a HExxH motif, ChiA represents a new class of peptidase that can degrade mammalian mucin-like proteins via a novel mechanism. We have identified four residues in ChiA that are essential for zinc binding in the peptidase active site (His506, His544, Asn547 and Gln595) and these likely coordinate a single zinc ion. These residues along with Asp504, Gln583 and Gln617 are also essential for peptidase activity. Examination of the ChiA-CTD structure suggests that His544, Asn547 and Gln595 are ligands for the zinc, with Asp504 the general base (Fig 8C). Active site zinc ligands are usually histidine, glutamate, aspartate or cysteine residues [55] but neutral residues such as asparagine and glutamine have been observed in other enzymes [56–58]. His506 packs against the N547 and H544 loop and may have a role in correctly structuring this region of ChiA-CTD. However, we cannot rule out the involvement of H544 binding zinc ions in this region. Gln583 and Gln617 have important roles in the optimal positioning of chitin for processing within the chitinase active site (Fig 2B). Lack of peptidase activity in ChiA-CTDGln583 and ChiA-CTDGln617 suggests that these residues are also involved in binding glycan motifs in mucin-like proteins. While Asp504 is located in an augmented α3 helix, His544, Asn547 and Gln595 are positioned within conserved GH18 secondary structure (S4 Fig). However, sequence alignment of L. pneumophila ChiA with other virulent bacterial chitinases, including the mucin degrading V. cholerae ChiA2 [59], does not show conservation of these residues and modelling of their tertiary structures using the Phyre2 server [23] also highlights significant differences within their chitin binding surfaces. This implies that other virulent bacterial chitinases either promote pathogenesis using an alternative mechanism or that the specific location of the peptidase active site is unique to each enzyme and shapes their glycan specificity and function. Mucins derived from the lung are not commercially available, but we have shown that ChiA has specificity for and can degrade MUC5AC purified from the porcine stomach, which is also a major mucin expressed in the human airway and lung [45]. MUC5AC is composed of T-antigen (Galβ1-3GalNAcαSer/Thr), core 2 (GlcNAcβ1-6(Galβ1–3)GalNAcαSer/Thr) and sialyl T-antigen (NeuAcα2-6(Galβ1–3)GalNAcαSer/Thr) core glycan structures [60]. While the T-antigen contains a linear array of carbohydrates, core 2 and sialyl T-antigen have branched structures. Our study suggests that the specificity of ChiA-CTD for O-glycosylated substrates is mediated by glycan recognition in the chitin binding groove, which then orientates the peptide backbone of the substrate towards the peptidase active site for proteolysis (Fig 8C). Furthermore, the MPD-2 site which we observed in the crystal structure of ChiA-CTD may also represent an additional glycan surface to accommodate branched glycan structures. MUC5AC is a gel forming mucin and our mucin penetration assay indicates that one role for ChiA in the lung is to facilitate bacterial penetration of the alveolar mucosa, which would increase access to host tissue. However, we have also demonstrated that the peptidase activity of ChiA-CTD is active against human C1-INH. This is a major glycoprotein in plasma that regulates the complement pathway through inactivating the proteases C1r, C1s and mannose-binding protease-associated serine protease 2 (MASP2) [61]. In Bordetella pertussis the secreted Vag8 protein has been shown to bind and release C1-INH from C1r, C1s and MASP2 [62] where these active proteases can then cleave C2 and C4 from the bacterial surface. This indicates that ChiA may also modulate host immune responses to L. pneumophila infection via evasion of the complement pathway. Secreted ChiA has been detected in bacterial culture supernatants and during L. pneumophila infection of cultured amoebae and human macrophages [17, 32]. We previously observed that in the early stages of macrophage infection ChiA and another type II substrate ProA are exported to the host cytoplasm and localize to the surface of the LCV [32]. The ability of ChiA to bind non-chitin substrates implies that during intracellular infection ChiA interacts with other specific host cytoplasmic glycoproteins and may modulate host pathways either through their requisitioning to the LCV or through their hydrolysis. In this study we have now shown that ChiA can also bind the L. pneumophila surface, through its N3 domain, although ProA does not. In other bacteria, type II substrates can associate with their outer membrane via acetylation of their N-terminus, interactions with other outer membrane proteins or through recognition of lipopolysaccharides [11, 33]. While the mechanism by which ChiA is targeted to the Legionella outer membrane is still unclear, this may be shared during localization of ChiA to the LCV, yet interactions that tether ProA to the LCV are clearly not also present on the L. pneumophila surface. Our examination of recombinant ChiA subdomains has revealed that the N-terminal domains and C-terminal chitinase/peptidase domain can all bind porcine stomach mucins and implies that host mucins can be sequestered and processed on the bacterial surface. However, we did not observe significant differences between mucin binding to L. pneumophila 130b wild-type or chiA mutant strains and this demonstrates that additional uncharacterized mucin binding proteins may be present on the bacterial surface. We also identified additional mucinase activity in the culture supernatants of both L. pneumophila 130b wild-type and chiA mutant strains, which implies that other yet to be identified mucinase enzymes are secreted by L. pneumophila, compatible with past bioinformatic assessments of the L. pneumophila genome [10]. Together this indicates that manipulation of the host mucosa is an important pathogenic mechanism of L. pneumophila. The multifaceted nature of ChiA makes it a highly versatile virulence factor of L. pneumophila and likewise a target for controlling L. pneumophila infection. As a surface associated protein ChiA is a promising vaccine target and our structural characterization may provide a platform to initiate vaccine development. Materials and methods Cloning, expression and purification Full-length ChiA (ChiA-FL; residues 1–762), minus the N-terminal periplasmic signal sequence, the ChiA N-terminal region (ChiA-NT; residues 1–417), the ChiA N1-domain (ChiA-N1; residues 1–140), the ChiA N2-domain (ChiA-N2; residues 138–299), the ChiA N3-domain (ChiA-N3; residues 285–417) the ChiA C-domain (ChiA-CTD; residues 419–762) and NttE (residues 1–269) were amplified from the genomic DNA of L. pneumophila strain 130b and cloned into the N-terminal His6-tagged vector pET-46 Ek/LIC (S4 Table). SslE (residues 67–1497), minus the N-terminal periplasmic signal sequence and mature SslE N-terminal proline-rich region, were amplified from the genomic DNA of E. coli strain W and cloned into the C-terminal His6-tagged vector pOPINE (S4 Table). These were transformed into E. coli SHuffle cells (New England Biolabs) and grown at 37°C in LB media with 100 μg/ml ampicillin. Expression was induced with 1 mM isopropyl-d-1-thiogalactopyranoside (IPTG) at an OD600nm of 0.6 and cells were harvested after growth overnight at 18°C. Cells were resuspended in 20 mM Tris–HCl pH 8, 200 mM NaCl, 5 mM MgCl2, 1 mg/ml DNase I, 5 mg/ml lysozyme, lysed by sonication and purified using nickel affinity chromatography. All samples were then gel filtered using a Superdex 200 column (GE Healthcare) equilibrated in 20 mM Tris–HCl pH 8, 200 mM NaCl. Nuclear magnetic resonance spectroscopy One-dimensional proton NMR experiments were performed at 25°C on 300 μM ChiA-NT, ChiA-N1, ChiA-N2, ChiA-N3 and ChiA-CTD samples in a buffer containing 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% D2O. Spectra were recorded on a Bruker Avance III 700 MHz (ChiA-N1, ChiA-N2, ChiA-N3, ChiA-CTD) or 800 MHz (ChiA-NT) spectrometer equipped with cryoprobes and processed within TopSpin (Bruker). Site-directed mutagenesis E543M chiA-CTD mutant was created using pET46chiA-CTD template DNA with a QuikChange II Site-Directed Mutagenesis Kit (Stratagene) (S4 Table). D504A, H506A, H544A, N547A, Q583A, Q595A, Q617A, E543M/D504A, E543M/H506A, E543M/H544A, E543M/N547A and E543M/Q595A chiA-CTD mutants were synthesized by Synbio Technologies and cloned into pET28b vector using NcoI and XhoI restriction sites (S5 Table). All resulting clones were verified by DNA sequencing and then expressed and purified as described for wild-type ChiA-CTD. Circular dichroism Far-UV CD spectra were measured in a Chirascan (Applied Photophysics) spectropolarimeter thermostated at 20°C. Spectra for wild-type ChiA-CTD and ChiA-CTDD504A, ChiA-CTDH506A, ChiA-CTDE543M, ChiA-CTDH544A, ChiA-CTDN547A, ChiA-CTDQ583A, ChiA-CTDQ595A, ChiA-CTDQ617A, ChiA-CTDE543M/D504A, ChiA-CTDE543M/H506A, ChiA-CTDE543M/H544A, ChiA-CTDE543M/N547A and ChiA-CTDE543M/Q595A mutants (0.05 mg/ml) in 10 mM Tris-HCl pH 8.0, 100 mM NaCl were recorded from 260 to 200 nm, at 0.5 nm intervals, 1 nm bandwidth, and a scan speed of 10 nm/min. Three accumulations were averaged for each spectrum. Chitinase activity assay Enzyme activity was determined using 4-Nitrophenol β-D-N,N’,N”-triacetylchitotriose (Sigma) as a substrate. All experiments were performed in triplicate. 10 μl of ChiA-FL, ChiA-NT, ChiA-CTD, ChiA-CTDE543M, ChiA-CTDQ583A or ChiA-CTDQ617A at 10 μg/ml in PBS were mixed with 90 μl of substrate at 0.4 mg/ml dissolved in 20 mM sodium acetate pH 4.8. These samples and a 300 μl standard (50 μM ρ-nitrophenol, 100 mM sodium carbonate) were incubated at 37°C for 30 min and then the ChiA reactions were quenched with the addition of 200 μl of 100 mM sodium carbonate. The release of the chromophore ρ-nitrophenol (pNP) was measured at 405 nm and ChiA samples were corrected for absorption in a control sample with added PBS instead of protein. 1 unit of activity per mg enzyme (U/mg) was defined as the release of 1 mmol of pNP/mg of ChiA/min. Chitin binding assay 250 μl ChiA-FL, ChiA-NT, ChiA-N1, ChiA-N2, ChiA-N3, ChiA-CTD, ChiA-CTDD504A, ChiA-CTDH506A, ChiA-CTDE543M, ChiA-CTDH544A, ChiA-CTDN547A, ChiA-CTDQ595A and BSA (Sigma) at 10 μM in 20 mM Tris-HCl pH 8.0, 200 mM NaCl were incubated with 50 μl chitin-resin (Sigma) and incubated whilst shaking for 30 min. The resin was washed three times with 500 μl of the same buffer and then proteins were eluted by incubating the resin in 250 μl of 8 M urea, 1% (w/v) SDS for 30 min whilst shaking. Protein samples prior to incubation with chitin-resin and the eluted protein/chitin-resin slurry were then analysed with SDS-PAGE. Eluted samples underwent an upward shift compared to the input samples due to the large differences in buffer conditions. Crystal structure determination Crystallization of ChiA CTD-domain (30 mg/ml) was performed using the sitting-drop vapour-diffusion method grown in 0.2 M ammonium acetate, 0.1 M Bis-Tris pH 5.5, 45% (v/v) 2-Methyl-2,4-pentanediol at 293K. Native crystals were flash cooled in liquid nitrogen and diffraction data were collected at 100K on beamline I04 at the Diamond Light Source (DLS), UK. Crystals were also soaked for 1 min in well solution containing 1.0 M NaI, flash cooled in liquid nitrogen and data were collected at 100K on beamline I02 at the Diamond Light Source (DLS), UK. Data were processed with XDS [63] and scaled with AIMLESS [64] using the XIA2 pipeline [65]. The structure of ChiA CTD-domain was determined with I-SIRAS. Twenty-one iodide sites were located in ChiA C-domain using SHELXD [66], and then phases were calculated using autoSHARP [67]. After automated model building with ARP/wARP [68], the remaining structure was manually built within Coot [69]. Refinement was carried out with REFMAC [70] using non-crystallographic symmetry (NCS) and translation-libration-screw (TLS) groups [71, 72], and 5% of the reflections were omitted for cross-validation. Processing and refinement statistics of the final model can be found in Table 1. SAXS data collection and analysis SAXS data were collected on beamline B21 at the Diamond Light Source (DLS), UK at 20°C. Full-length ChiA in 20 mM Tris–HCl pH 8, 200 mM NaCl were measured at 4, 2, 1 and 0.5 mg/ml concentrations, after gel filtration using a Superdex 200 column (GE Healthcare), over a momentum transfer range of 0.0048.5 ppm) correspond to highly ordered backbone amides present in secondary structure elements. https://doi.org/10.1371/journal.ppat.1008342.s001 (TIF) S2 Fig. Circular dichroism (CD) spectra of ChiA-CTD constructs. The negative bands between ~210 to ~ 220 nm and positive band at 200 nm is indicative of a mixed α/β protein fold. The spectra for wild-type (WT) ChiA-CTD and mutants are in essence identical and demonstrates that these mutations do not perturb the structure of the CTD domain. https://doi.org/10.1371/journal.ppat.1008342.s002 (TIF) S3 Fig. MPD bound to ChiA-CTD. Electrostatic surface potential representation of ChiA-CTD with two molecules of MPD shown as spheres (MPD1: 4S enantiomer; MPD2: 4R enantiomer). Each binding site is expanded and the ρA weighted electron density maps contoured at 1.0 r.m.s. are shown. https://doi.org/10.1371/journal.ppat.1008342.s003 (TIF) S4 Fig. Sequence alignment of Legionella pneumophila ChiA-CTD and Bacillus cereus ChiNCTU2. Secondary structure elements of ChiNCTU2 and ChiA-CTD are shown above and below, respectively (green rectangle: α-helix; gold arrow: β-strand). Amino acid identities and similar residues are indicated by background shading in cyan and yellow, respectively. Catalytic chitinase residues and chitin binding residues in ChiNCTU2 are indicated with red and blue filled circles, respectively. Mucinase active site residues in ChiA-CTD are shown as open red circles. https://doi.org/10.1371/journal.ppat.1008342.s004 (TIF) S5 Fig. Superposition of ChiA-CTD tertiary homologs. L. pneumophila ChiA-CTD is green, Bacillus cereus ChiNCTU2 is purple (PDB ID code 3n18) [27], Bacillus anthracis Chi36 is red (PDB ID code 5kz6, Chromobacterium violaceum ChiA is yellow (PDB ID code 4tx8) and Streptomyces coelicolor ChiA is blue (PDB ID code 3ebv). Augmented loop and helical structures in L. pneumophila ChiA-CTD are annotated. https://doi.org/10.1371/journal.ppat.1008342.s005 (TIF) S6 Fig. SAXS analysis of ChiA-FL. (A) Comparison of scaled scattering curves of ChiA-FL at 0.5 mg/ml (black), 1.0 mg/ml (red) and 2.0 mg/ml (teal) to highlight aggregation at concentrations above 1.0 mg/ml. (B) Experimental scattering curve of ChiA-FL (black open circles). Inset: Guinier Region (orange open circles) and linear regression (black line) for Rg evaluation. (C) Shape distribution [P(r)] function derived from SAXS analysis for ChiA. (D) Kratky, (E) Kratky-Debye and (F) Porod-Debye plots indicate that ChiA is a highly dynamic particle in solution. https://doi.org/10.1371/journal.ppat.1008342.s006 (TIF) S7 Fig. Antibody binding to recombinant ChiA fragments. ELISA analysis of anti-ChiA antibodies binding to either full-length ChiA (FL), the N-terminal domain of ChiA (NT), and the C-terminal domain of ChiA (CTD) (left panel) or the ChiA N-terminal subdomain 1 (N1), subdomain 2 (N2), and subdomain 3 (N3) (right panel). All values represent the mean and standard deviation from triplicate wells. https://doi.org/10.1371/journal.ppat.1008342.s007 (TIF) S8 Fig. L. pneumophila growth on mucin supplemented media. WT and chiA mutant bacteria were grown from a starting OD660 of 0.3 in chemically defined medium in the presence of porcine mucin II at the indicated concentrations. At 0 h, 8 h and 24 h, bacterial numbers were determined by plating for CFU. N = 3. Representative graph shown above as mean and standard deviation of technical replicates in triplicate. Two other experiments showed the same trends, with no significant difference between mutant or mucin effect. https://doi.org/10.1371/journal.ppat.1008342.s008 (TIF) S9 Fig. Reverse ITC titration. Titration of ChiA-CTD (syringe) into Zn2+ (cell) to assess heat generation during the dilution of ChiA-CTD. No significant heat generation was observed. https://doi.org/10.1371/journal.ppat.1008342.s009 (TIF) S10 Fig. Chitin-resin pull down with ChiA mutants. SDS-PAGE gels loaded with ChiA-CTD mutants or BSA control either before incubation with chitin beads (L) or after elution from the beads (B). Eluted samples undergo an upward shift compared to the input sample due to differences in buffer conditions. Data is representative of three independent repeat experiments. https://doi.org/10.1371/journal.ppat.1008342.s010 (TIF) S11 Fig. Mucin binding of ChiA-CTD mutants. ELISA analysis of binding between immobilised type II or III mucin extracts and His-tagged wild-type ChiA-CTD (WT), ChiA-CTD mutants (D504A, H506A, E543M, H544A, N547A, Q583A, Q595A, 617A) and controls (SslE, NttE). Anti-His-tag antibody conjugated to HRP was used to measure OD450 nm values. BSA-coated wells were used as controls. Data represent the mean and standard deviation for triplicate experiments. *, P < 0.001; verses control empty well by two-tailed Student’s test. https://doi.org/10.1371/journal.ppat.1008342.s011 (TIF) S12 Fig. Controls for detection of proteins bound to L. pneumophila surface. Whole cell ELISA of L. pneumophila wild-type 130b (WT) and mip mutant NU203 (mip) detected with Mip-specific antiserum, and L. pneumophila wild-type 130b (WT) and L. pneumophila lysed by freeze-thaw lysed (FT-WT) probed with an ICDH-specific antiserum that recognizes a cytosolic L. pneumophila protein. Data represent the mean and standard deviation. *, P < 0.001; verses WT by two-tailed Student’s test. https://doi.org/10.1371/journal.ppat.1008342.s012 (TIF) S1 Table. Tertiary structure predictions of ChiA N-terminal subdomains. https://doi.org/10.1371/journal.ppat.1008342.s013 (PDF) S2 Table. SAXS structural parameters. https://doi.org/10.1371/journal.ppat.1008342.s014 (PDF) S3 Table. SAXS ensemble optimization parameters. https://doi.org/10.1371/journal.ppat.1008342.s015 (PDF) S4 Table. Primers used in this study. https://doi.org/10.1371/journal.ppat.1008342.s016 (PDF) S5 Table. Synthetic genes. https://doi.org/10.1371/journal.ppat.1008342.s017 (PDF) Acknowledgments We thank the beamline scientists at I02, I04 and B21 of the Diamond Light Source for support during data acquisition and Dr Ben Stieglitz (Queen Mary University of London) for providing ITC support. TI - Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation JF - PLoS Pathogens DO - 10.1371/journal.ppat.1008342 DA - 2020-05-04 UR - https://www.deepdyve.com/lp/public-library-of-science-plos-journal/structure-and-functional-analysis-of-the-legionella-pneumophila-volhn3NG2e SP - e1008342 VL - 16 IS - 5 DP - DeepDyve ER -