The PE_PGRS Proteins of Mycobacterium tuberculosis Are Ca2+

Aug 2, 2016 - *CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. Telephone: +91-40-27192924...
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The PE_PGRS proteins of Mycobacterium tuberculosis are Ca binding mediators of host-pathogen interaction 2+

Veena C. Yeruva, Apoorva Kulkarni, Radhika Khandelwal, Yogendra Sharma, and Tirumalai R Raghunand Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00289 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 2, 2016

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A. Title. The PE_PGRS proteins of Mycobacterium tuberculosis are Ca2+ binding mediators of hostpathogen interaction B. Funding Source Statement. This work was supported by grants from the Council of Scientific and Industrial Research (CSIR) Government of India to TRR (BSC104-SPLenDID) and to YS (BSC0208BIOAGE). C. Byline. Veena C Yeruva*, Apoorva Kulkarni, Radhika Khandelwal, Yogendra Sharma and Tirumalai R Raghunand* CSIR - Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India *

To whom correspondence should be addressed. Raghunand R. Tirumalai, CSIR - Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. Tel: +91-40-27192924; Fax: +91-40-27161591; E-mail: [email protected]. Veena C Yeruva, CSIR - Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. Tel: +91-40-27192930; Fax: +91-40-27161591; E-mail: [email protected]

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Abstract The phenomenal success of Mycobacterium tuberculosis (M.tb) as a pathogen is primarily based on its ability to modulate host immune responses. The genome of M.tb encodes multiple immunomodulatory proteins, including several members of the multigenic PE_PPE family of which the PE_PGRS proteins are a subset. Curiously, 56 of the 61 PE_PGRS proteins contain multiple copies of the glycine-rich sequence motif GGXGXD/NXUX a nona peptide sequence predicted to bind Ca2+, but the functional significance of these motifs remains a mystery. Here we provide evidence via isothermal titration calorimetry, 45Ca blotting, fluorescence and circular dichroism spectroscopy, that Ca2+ binds to the PE_PGRS proteins - PE_PGRS33 (Rv1818c) (10 motifs) and PE_PGRS61 (Rv3653) (1 motif). Ca2+ was observed not to bind to PE_PGRS8 (Rv0742), which lacks nona peptide motifs. Using recombinant Mycobacterium smegmatis strains expressing Rv1818c and Rv3653 and the THP-1 macrophage model of infection, we show that the two proteins mediate Ca2+ dependent up-regulation of the anti-inflammatory cytokine IL-10, events critical to the pathogenesis of M.tb. Both Rv1818c and Rv3653 interact with TLR2 in a Ca2+ dependent manner, providing a novel mechanistic basis for their immunomodulatory effects. Mutations in the nona peptide motif of Rv3653 led to compromised Ca2+ binding, validating the functional criticality of this motif. This study demonstrates for the first time, not only their Ca2+ binding properties, but also an essential role for Ca2+ in the functioning of the M. tb PE_PGRS proteins, opening up the possibility of developing novel anti-TB therapeutics that inhibit Ca2+-PE_PGRS binding.

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List of Abbreviations: M.tb, Mycobacterium tuberculosis; TLR, Toll-like receptor; PE_PGRS, Proline Glutamate Polymorphic GC-rich Repetitive Sequence; IL-10, interleukin-10; ITC, Isothermal Titration Calorimetry; CD, Circular Dichroism; RTX, repeat in toxin.

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Ca2+ is a universal secondary messenger affecting major cellular processes in eukaryotes by binding to proteins with varied affinities in both extracellular and intracellular environments (1, 2). Although prokaryotic equivalents of eukaryotic Ca2+ binding proteins remain virtually unexplored, emerging evidence suggests the involvement of Ca2+ in diverse cellular processes in bacteria (3-6). Several reports suggest that bacteria are able to maintain intracellular Ca2+ homeostasis, indicating its importance in the regulation of prokaryotic physiology (7, 8). Pathogens such as Mycobacterium tuberculosis (M.tb), the causative agent of human tuberculosis, possess the ability to alter Ca2+ levels in macrophages to favour their own survival (9), implying a link between Ca2+ and the innate immune response. Ca2+ signaling has been observed to be critical for the maturation of phagosomes containing mycobacteria (10-12), their maturation and their acidification can be artificially restored by raising the levels of cytosolic Ca2+ using Ca2+ ionophores (10). Pathogenic mycobacteria have the ability to survive in the phagosomal compartment and inhibit lysosomal delivery by intracellular Ca2+ chelation, which can be mimicked by pharmacological inhibition of calmodulin, CaMKII, or sphingosine kinase 1(10-12). M.tb infection in macrophages has also been observed to cause a persistent rise in the concentration of intracellular Ca2+ ions, which activates the Ca2+-dependent protein phosphatase 2B calcineurin, leading to a block in phagosome-lysosome fusion, allowing bacillary survival. This activation was demonstrated to depend on the recruitment of coronin 1, a leucocyte-specific protein, to the mycobacterial phagosome (13). While the survival of M.tb inside the host is intimately linked to levels of intracellular Ca2+, the role of extracellular Ca2+ in M.tb pathogenesis has not been investigated thus far. This is likely to involve Ca2+ binding proteins localised on the bacillary surface, which mediate Ca2+ dependent interactions at the hostpathogen interface. The multigenic PE_PPE family (named after the conserved Proline-Glutamate and Proline-Proline-Glutamate residues at their N-termini) which comprise about 10% of the coding potential of the M.tb genome have been demonstrated to contain several surface associated proteins implicated in host immune evasion (14-18). Within this family are 61 PE_PGRS (Polymorphic GC-rich Repetitive Sequence) members with a conserved N-terminus of ∼110 aa, and a C-terminal region comprised of glycine-rich repeat regions. PE_PGRS33, encoded by Rv1818c is the best studied protein of the PE_PGRS family (19-22). It has been observed to trigger macrophage cell death by inducing secretion of pro-inflammatory cytokines (22, 23) and the activation of pro-apoptotic or pro-necrotic signals involving mitochondria (19, 24, 25). Most recently, PE_PGRS33 was observed to mediate entry of M.tb in to macrophages through its interaction with TLR2 (26). In addition, the PE_PGRS family also encodes genes necessary for M.tb multiplication and persistence within macrophages. PE_PGRS62 (Rv3812) is involved in supporting virulence via the inhibition of phagosome maturation and iNOS expression, phenotypes possibly linked to effects on bacterial cell wall composition (27, 28). Also, PE_PGRS30 (Rv1651c) was found to be involved in modulation of mycobacterial growth and suppression of the proinflammatory immune response (29, 30). Interestingly, it was found that of the 61 PE_PGRS proteins, 56 are predicted to contain repeats of the nona peptide GGXGXD/NXUX (where X is any aa and U is an unpolar/ large hydrophobic residue) which possibly constitute a Ca2+ binding motif similar to what is known as a parallel β-roll or parallel β-helix (31). This motif was first identified in the Ca2+ -binding RTX (Repeat in Toxin) toxins secreted by many Gram-negative bacteria (32, 33). Consequently, it is probable that these nona peptide motifs may confer a Ca2+ -binding property to the PE_PGRS proteins, with potential implications in the context of host-M.tb interactions. We hypothesise that Ca2+ dependent interactions between the PE_PGRS proteins of M.tb and host immune cells are crucial to the pathogenesis of M.tb, and demonstrate for the first time a novel role for extracellular Ca2+ in M.tb - macrophage interactions. By detailed biophysical characterisation of the PE_PGRS proteins Rv1818c and Rv3653, we unequivocally show that Ca2+ binds to these proteins and induces moderate conformational changes. In addition, we provide evidence that these proteins mediate Ca2+-dependent immunomodulatory signaling via TLR2. Amino acid changes in the Ca2+ binding nona peptide motif are poorly tolerated and lead to decreased Ca2+ -binding affinity. Our results reveal an unknown facet in the functioning of this important class of proteins in the pathophysiology of M.tb, and

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assume significance in the development of therapeutic strategies targeting the Ca2+ -binding sites of the PE_PGRS proteins. Experimental procedures Bacterial strains, media and growth condition Mycobacterium smegmatis mc2 155 was cultured in Middlebrook 7H9 broth or Middlebrook 7H10 agar (Difco), supplemented with albumin dextrose complex (5 g BSA, 2 g glucose and 0.85 g NaCl/l), 0.05% v/v Tween 80 (Sigma-Aldrich) and 0.5% v/v glycerol (Sigma-Aldrich). E. coli strains were cultured in Luria–Bertani medium (LB). Both E. coli and mycobacteria were grown at 37 °C with shaking. Ampicillin (200 μg/ml) and Kanamycin (50 μg/ml for E. coli and 15 μg/ml for mycobacteria) were used for antibiotic selection. DNA manipulations Protocols for DNA manipulations, including plasmid DNA preparation, restriction endonuclease digestion, agarose gel electrophoresis, isolation and ligation of DNA fragments, and E. coli transformation were performed as described (34). PCR amplifications were carried out according to the manufacturer’s specifications. Each of the 30 cycles was carried out at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min/1000 bp, followed by a final extension cycle at 72 °C for 10 min. DNA fragments used for cloning reactions were purified by a Qiagen gel extraction kit according to the manufacturer’s specifications. Purification of recombinant proteins For their cloning and expression, the ORFs corresponding to Rv1818c, Rv3653 (and its mutant alleles) and Rv0742 were amplified from M.tb H37Rv genomic DNA using gene-specific primers (See Table S1) and cloned along with with C-terminal 6xHIS tags between the BamHI and EcoRI sites of pGEX6P1 and transformed into E. coli BL21(DE3). The proteins expressed in this system, therefore contain both a Cterminal 6xHis tag as well an N-terminal GST tag. This strategy became necessary since we were unable to observe over expression of proteins solely as 6xHis tagged fusions. Also, since GSH affinity based purification resulted in suboptimal yields, adding the 6xHis tag became essential in order to purify these proteins to homogeneity via Ni2+ -NTA affinity purification. Transformant cultures were induced at the logarithmic phase of growth with 0.5 mM IPTG for 3 hours at 37° C. The cell pellets were resuspended in binding buffer containing 50 mM Tris pH 7.5, 100 mM KCl, 10 mM imidazole, 1 mg/ml lysozyme and 250 mM PIC (protease inhibitor cocktail) and sonicated with 6 sec on and 3 sec off pulses for 30 min at 4 °C, following which the cell lysates were centrifuged at 14000 rpm for 15 min. Rv1818c (66 kDa) and Rv0742 (41.5 kDa) were purified from the inclusion body fractions by solubilisation using 50 mM TrisHCl pH 8.9 and 100 mM KCl containing 3.5 M Urea. The solubilized inclusion bodies were refolded on a pre-equilibrated Ni2+ -NTA affinity purification column by setting a gradient between 3.5 M - 0 M urea in the above buffer and eluted using 500 mM imidazole. Rv3653 (41.8 kDa), Rv3653 N166A and Rv3653 G168S were purified from the soluble fractions using pre-equilibriated Ni2+ NTA affinity columns. After performing gradient washing (50 mM Tris pH 7.5, 100 mM KCl) containing imidazole (20 mM and 40 mM), the proteins were eluted in a buffer containing 50 mM Tris pH 7.5, 100 mM KCl and 500 mM imidazole. Protein concentrations were estimated using BCA. The fractions containing protein were concentrated by centrifugal concentrators with a 30 kDa molecular mass cut -off membrane. The concentrated protein was finally purified by Superdex-75 gel filtration column on a Bio-Rad Duo-Flow purification system (hereby referred to as Native Protein (NP)). For all Ca2+ binding studies, this protein preparation was treated with 10 µM EDTA for 15 min at RT, followed by chelex (Chelex 100 resin, Sigma-Aldrich) buffer exchange (50 mM Tris HCl pH 7.5, 100 mM KCl) (hereby referred to as the apo form of the protein).

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Fluorescence Spectroscopy Intrinsic tryptophan fluorescence emission spectra of Rv1818c were recorded on an F-4500 fluorescence spectrofluorimeter (Hitachi Inc., Japan) with 0.1 mg/ml protein in 50 mM Tris-HCl (pH 7.5) and 100 mM KCl. The spectra were recorded from 300 to 450 nm at an excitation wavelength of 295 nm in the correct spectrum mode of the instrument using excitation and emission band passes of 5 nm each. Changes in surface hydrophobicity of Rv1818c, Rv3653 and Rv0742 (0.1 mg/ml) in response to varying concentrations of CaCl2 (10 µM to 5 mM) were monitored using 8-anilinonaphthalene-1-sulfonic acid (ANS) (100 µM) as a probe. The samples were excited at 365 nm, and emission spectra were recorded between 400 nm and 650 nm. The spectra were corrected for equal concentrations of ANS in the buffer. Circular Dichroism (CD) spectroscopy Far- UV CD spectra of Rv1818c and Rv3653 were recorded on a Jasco-815 spectropolarimeter using 0.01 cm path length cuvettes at room temperature. The change in the protein conformation upon binding Ca2+ was studied by titrating 0.1 mg/ml protein solutions with aliquots of CaCl2 ranging in concentration from 0 to 5mM. Isothermal Titration Calorimetry (ITC) Ca2+ binding isotherms of proteins were determined using a Microcal VP-ITC (Microcal Inc., USA). The protein samples and Ca2+ (100 µM Rv1818c with 10 mM CaCl2, 60 µM Rv3653 with 5 mM CaCl2 and 50 µM Rv0742 with 5 mM CaCl2) were prepared in Chelex-purified 50 mM Tris, pH 7.5 and 100 mM KCl buffer. The experiment was carried out by loading protein in the cell (1.4 ml) and CaCl2, in the syringe at 30 °C. Data fitting was performed using the Origin software (version 7.0) supplied by Microcal after subtraction with the appropriate buffer blank. 45

CaCl2 Overlay Assay Ca2+ binding to Rv1818c, Rv3653 and Rv0742 was evaluated by 45Ca membrane overlay using a modification of a protocol described previously (35). Briefly, 20 µg Chelex buffer exchanged Rv1818c and Rv3653 were transblotted onto a PVDF membrane, which was then incubated with 1 μCi/ml 45CaCl2 (BRIT, Mumbai) in the above buffer for 1 h. BSA fraction V (20 µg) and purified Glutathione S transferase (GST) were used as negative controls. Caldendrin, a neuron-specific Ca2+-binding protein (36) was used as a positive control. The membrane was washed with 50% ethanol for 15 min, air-dried and subjected to autoradiography. Densitometric quantitation of the bands was performed using Image J software. The pixel intensity of each 45Ca overlay band was normalized to its corresponding Ponceau stained loading control. Proteinase K sensitivity assay and sub-cellular localization To evaluate their surface localisation in M. smegmatis, ORFs of M.tb Rv1818c, Rv3653 and Rv0742 were amplified from M.tb H37Rv genomic DNA using gene-specific primers (Table S1) and cloned between the BamHI and EcoRI sites of pJEX55 as C-terminal myc fusions. The recombinant plasmids were transformed into M. smegmatis and the transformants cultured and harvested at their logarithmic phase of growth. The pellets were washed and resuspended in PBS following which, each sample was divided into two identical aliquots and incubated with and without 100 μg/ml Proteinase K (Sigma) at 37 °C for 30 min. The reaction was stopped by the addition of 2 mM EGTA and the samples were washed once in PBS and re-suspended in PBS containing SDS-PAGE loading buffer. Both samples were electrophoresed using denaturing PAGE and the fusion proteins detected by western blotting with anti c-myc monoclonal antibodies (sc40, Santa Cruz). Fractionation of M. smegmatis strains expressing c-myc-tagged Rv1818c, Rv3653 & Rv0742, and immunoblotting to detect the fusion proteins, were performed as described (18).

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Expression of Rv1818c, Rv3653 and Rv0742 in M. smegmatis To functionally characterize M.tb Rv1818c, Rv3653 and Rv0742 their ORFs were amplified from M.tb H37Rv genomic DNA using gene-specific primers (Table S1), cloned between the BamHI and EcoRI sites of pMV261 (37), and transformed into M. smegmatis. For gene expression analyses total RNA was isolated from recombinant strains at their exponential phase of growth using TRIzol reagent (Invitrogen). Following DNAse I treatment, cDNA synthesis was performed using Superscript II (Invitrogen) and subsequently used as a template for PCR amplification using gene-specific primers. Real-time PCR Analysis To determine the expression profiles of Rv1818c, Rv3653 and Rv0742 in recombinant strains of M. smegmatis expressing pMV261-Rv1818c pMV261-Rv3653 and pMV261-Rv0742 as a function of growth, cells were harvested at the 6, 12, 24, 48 and 72 h time points and total RNA isolated from each culture using TRIzol reagent as per the manufacturer’s protocol. Following treatment with RNAse-free DNAse I, cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio-Rad) and subsequently used as a template for SYBR green based PCR amplification using Rv1818c, Rv3653and Rv0742 specific primers to generate 200 bp amplicons (Table S1). Gene-specific transcript levels were normalised to the M. smegmatis sigA transcript in each sample. The relative fold change in transcript levels at each time point was calculated with respect to the levels at 6 h which was assigned a value of 1. To quantify iNOS transcript levels, total RNA was isolated from infected macrophages at 24, 48 and 72 h post infection using TRIzol reagent and processed for real-time PCR as described above. The levels of each mRNA were normalised to the transcript levels of β-actin. Relative fold changes at each time point were calculated with reference to macrophages infected with M. smegmatis expressing the empty vector, which was assigned a value of 1. In vitro growth kinetics To examine their growth patterns, recombinant M. smegmatis strains expressing pMV261-Rv1818c, pMV261-Rv3653, pMV261-Rv0742 and the empty vector, were grown until late exponential phase and diluted to an OD of 0.2. Growth curves were generated by plotting CFU counts at different time points plotted against time. Macrophage infection and Ca2+ perturbation THP-1 macrophages were cultured at 37 °C in 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) Fetal bovine serum, 2 g/l sodium bicarbonate and antibiotics (60 μg/ml penicillin G sodium, 50 μg/mL streptomycin sulphate, and 30 μg/mL gentamycin sulphate). THP-1 monocytes were seeded at a density of 2x106 / well and differentiated using 5 ng/ml of PMA for 24 h, and infected 72 h later. To remove extracellular Ca2+, the macrophages were incubated in RPMI medium supplemented with 1 mM ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′ -tetra acetic acid (EGTA, pH 7.3), (designated as RPMI-EGTA) for 30 min at 37 °C. To standardise the conditions for this analysis, we examined the effect of varying concentrations of EGTA (1, 2, 5 and 10 mM) on the adherence of THP-1 macrophages as a function of time (30 min, 1 h, 6 h and 24 h). Only at 30 min time point at a concentration of 1 mM EGTA were the cells observed not to detach from their substratum, leading us to choose these conditions for the experiment. For Ca2+ supplementation, differentiated THP-1 cells were incubated in 2 mM CaCl2 solution (Sigma-Aldrich) for 30 min at 37°C. Exponentially growing bacterial cultures were pelleted, washed and resuspended in RPMI medium (Untreated control, designated as UT in Figures 5A, B and 7B) / RPMIEGTA / RPMI-CaCl2 (without antibiotics) to an OD of 1.0 and incubated for 15 min at 37 °C. For treatment with EGTA-CaCl2, initially both macrophages and bacilli were treated with 1 mM EGTA, incubated for 15 min and subsequently washed three times with 1X HBSS and then incubated with CaCl2 for 30 min at 37 °C. Single cell suspensions of recombinant M. smegmatis strains were obtained by passing cultures 5-6 times through 26½ gauge needles. Bacillary viability was assessed at each step by performing CFU counts. Equal numbers of each strain were used to perform infections at an MOI of

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1:100. After incubation with bacteria for 2 h, cells were washed with 1X HBSS, and subsequently with complete RPMI, complete RPMI-EGTA or RPMI-CaCl2 containing gentamycin, added to kill extracellular bacteria. In each experiment, a sample of macrophages infected with M. smegmatis expressing the empty vector was included as the control. Cytokine Assays Levels of IL-10 in the culture supernatants of infected THP-1 macrophages were quantitated by a two-site sandwich EIA (Becton Dickinson OptEIA) as per the manufacturer’s protocol. Construction of mutant alleles of Rv3653 The point mutants of Rv3653 - N166A and G168S, were constructed by site-directed mutagenesis using the plasmid pGEX6p1-Rv3653-6xHIS as a template. To generate these mutants, two-step PCR amplification was carried out using overlapping primers (Table S1) with site specific substitutions followed by DpnI digestion O/N at 37 °C. The digested products were transformed into E. coli DH5α and transformants processed for plasmid isolation. Mutants were confirmed by plasmid sequencing using vector specific forward and reverse primers. Receptor interaction assay To isolate the membrane fraction for receptor identification, PMA differentiated THP -1 cells were resuspended in cell lysis buffer (50 mM Tris, pH 8.9, 150 mM KCl, protease inhibitor cocktail), briefly sonicated, and centrifuged at 11,000 g for 1 h and the pellet resuspended in lysis buffer containing 1% CHAPS. For the co-immunoprecipitation experiments, 20 µg of native protein (NP) purified from E. coli, apo (no Ca2+) and 10 mM Ca2+ saturated Rv1818c-GST and Rv3653-GST along with mutant alleles N166A and G168S were mixed individually with 500 µg of the cell membrane fractions, and the volume was made up to 500 µL with lysis buffer. The reactions were rocked overnight at 4 °C, following which pull downs were performed with anti-TLR2 antibodies (Abcam). Prior to antibody addition, 50 µL of each sample was aliquoted as a loading control. Protein A agarose beads were then added to each sample and incubated overnight at 4 °C. Following three washes with Tris-KCl buffer pH 8.9, the beads were boiled in 20 µL of sample buffer and subjected to SDS-PAGE, and the co-immunoprecipitated proteins were detected with an anti-GST antibody (Qiagen). Equal loading of the protein and cell membrane mix was assessed by SDS-PAGE followed by Coomassie staining. Results Ca2+ binds to the PE_PGRS proteins with micromolar affinities. The number of predicted nona peptide repeats in the PE_PGRS proteins range from 1 (in Rv0754, Rv0980c, Rv1983 and Rv3653) to 77 (in Rv3345c), with 10 repeats being the most commonly occurring number (10 PE-PGRS proteins) (31). To investigate the binding of Ca2+ to the PE_PGRS proteins via these motifs, we selected Rv1818c which contains ten predicted motifs, and Rv3653, with one predicted motif (Figure 1) as candidate representatives of this protein family, and examined their Ca2+ -binding affinities and stoichiometries by ITC. Our analyses indicate that Ca2+ binds to the two proteins with varying affinities and modes of binding (Table 1). Ca2+ binding to Rv1818c was observed to be endothermic and enthalpically favourable, as illustrated by the negative sign of the algebraic sum of enthalpy change (Figure 2A). The thermogram best fits to a sequential five binding site model, the global dissociation constant being in the micromolar range (9.9 µM) (Table 1). On the other hand, Ca2+ binding to Rv3653 is exothermic and enthalpically driven, as indicated by the negative enthalpy change in the binding isotherms (Figure 2B) which best fits to a sequential one binding site model (kd = 34 μM) (Figure 2B Table 1). Ca2+ titration to Rv0742 (41.5 kDa) lacking a nona peptide motif (Figure 1) leads to precipitation. We further assessed the binding of Rv1818c and Rv3653 to Ca2+ using 45CaCl2 overlay assay. As shown in Figure 2C, both Rv1818c and Rv3653 showed significant Ca2+ binding whereas Rv0742 failed to bind Ca2+, with its band matching to

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those of the negative controls GST and BSA (35). These observations clearly illustrate the Ca2+ binding property of Rv1818c and Rv3653. Binding of Ca2+ to the PE_PGRS proteins Rv1818c and Rv3653 changes their conformation. Having established Ca2+ binding to the test proteins, we proceeded to examine the Ca2+ dependent changes in their surface hydrophobicity using ANS fluorescence. ANS binds to Rv1818c with an emission maximum at 485 nm, and a mild increase in the fluorescence intensity of ANS was observed upon Ca2+ addition (Figure 3A). This suggests a possible reorientation of side chains resulting in enhanced ANS binding; no such change was observed on titration of the protein with Mg2+. However, the addition of Ca2+ to the Mg2+-saturated protein led to a similar increase in ANS fluorescence (Figure 3B) implying a Ca2+ specific change in hydrophobicity. Although, the addition of Ca2+ did not alter the surface hydrophobicity of Rv3653, (Figure 3C), the addition of Ca2+ to Mg2+-saturated protein resulted in a minor but noticeable increase in fluorescence intensity (Figure 3D) indicating the specificity of Ca2+ binding to this protein. Consistent with its inability to bind Ca2+, we observed no changes in fluorescence of ANS mixed with Rv0742 with either Ca2+ (Figure 3E) or on addition of Ca2+ to Mg2+-saturated Rv0742 (Figure 3F). As expected, GST as tag did not contribute to conformational changes in the test proteins (Figure S1A and B). Given that Ca2+ binding led to noticeable changes in their surface properties, we used far-UV CD to monitor Ca2+ dependent secondary structural changes in Rv1818c and Rv3653. The far-UV CD spectrum of Rv1818c in the apo form shows an unusual spectrum with a broad minimum at 225 nm (apparently due to n-π* transition), with a cross over point at ~210 nm, followed by a weak positive signal. This spectral feature indicates a conformation distinct from the RTX motif proteins. The addition of Ca2+ shifts the positive ellipticity (near 200 nm) towards the negative, resulting in a change in the cross over point to ~205 nm. This causes a minor shift in the 225 nm minimum to 222 nm (Figure 4A). These changes to the spectra are more obvious upon further addition of Ca2+ (1 mM), which results in a drastic shift in the polarity at