Clostridium difficilehas a single sortase, SrtB, that can be inhibited by small-molecule inhibitors
© Donahue et al.; licensee BioMed Central Ltd. 2014
Received: 6 June 2014
Accepted: 12 August 2014
Published: 31 August 2014
Bacterial sortases are transpeptidases that covalently anchor surface proteins to the peptidoglycan of the Gram-positive cell wall. Sortase protein anchoring is mediated by a conserved cell wall sorting signal on the anchored protein, comprising of a C-terminal recognition sequence containing an "LPXTG-like" motif, followed by a hydrophobic domain and a positively charged tail.
We report that Clostridium difficile strain 630 encodes a single sortase (SrtB). A FRET-based assay was used to confirm that recombinant SrtB catalyzes the cleavage of fluorescently labelled peptides containing (S/P)PXTG motifs. Strain 630 encodes seven predicted cell wall proteins with the (S/P)PXTG sorting motif, four of which are conserved across all five C. difficile lineages and include potential adhesins and cell wall hydrolases. Replacement of the predicted catalytic cysteine residue at position 209 with alanine abolishes SrtB activity, as does addition of the cysteine protease inhibitor MTSET to the reaction. Mass spectrometry reveals the cleavage site to be between the threonine and glycine residues of the (S/P)PXTG peptide. Small-molecule inhibitors identified through an in silico screen inhibit SrtB enzymatic activity to a greater degree than MTSET.
These results demonstrate for the first time that C. difficile encodes a single sortase enzyme, which cleaves motifs containing (S/P)PXTG in-vitro. The activity of the sortase can be inhibited by mutation of a cysteine residue in the predicted active site and by small-molecule inhibitors.
KeywordsClostridium difficile Sortase Cysteine protease Fluorescence resonance energy transfer (FRET) Enzyme kinetics Enzyme inhibitors
Sortases are membrane-bound cysteine transpeptidases that anchor surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. Surface proteins anchored via sortases are often essential virulence factors important in colonization and invasion, evasion of the host immune system, and nutrient acquisition. The sorting process is mediated by a conserved C-terminal cell wall sorting signal on the anchored protein, comprised of a C-terminal recognition sequence (often LPXTG, where X is any amino acid), followed closely by a hydrophobic transmembrane domain and a positively charged tail . A conserved catalytic cysteine residue of the sortase cleaves the LPXTG motif of the polypeptide between the threonine and glycine residues and covalently attaches the protein to the peptidoglycan -.
There are six described sortase families, A-F, that share amino acid similarity . All catalyze similar transpeptidation reactions, but recognize different substrate motifs and serve different functions within the cell. Class A sortases (SrtA), such as the prototypical Staphylococcus aureus Sortase A (SaSrtA), are considered housekeeping sortases as they are capable of anchoring many functionally distinct proteins to the cell wall. SaSrtA, which recognizes an LPXTG motif, is responsible for anchoring a variety of surface proteins involved in adherence and immune response evasion, and is essential for virulence in animal models ,. SrtA orthologues have been found in the genomes of almost all Gram-positive bacteria ,-. Class B sortases are functionally different from class A in their substrate specificity. In S. aureus and B. anthracis, the sortase B gene (srtB) is part of an iron-regulated locus isd (iron-responsive surface determinant) responsible for heme-iron transport, and anchors the iron transporter protein, IsdC, by recognizing an NPQTN motif ,. Though mutating srtB has no effect on establishing infection, SaSrtB is required for persistence of the bacterium in mice .
Clostridium difficile, an anaerobic Gram-positive, spore-forming bacillus, is the leading cause of hospital-acquired infectious diarrhea in North America and Europe. Infection with C. difficile can result in a range of clinical presentations, from mild self-limiting diarrhea to the life-threatening pseudomembranous colitis (PMC), known collectively as C. difficile infection (CDI) . MLST studies have identified that the C. difficile population structure forms at least five distinct lineages that are all associated with CDI -. Complications of severe CDI can lead to toxic megacolon, bowel perforation, sepsis and death in up to 25% of cases . Broad-spectrum antibiotic usage is the greatest risk factor for development of CDI due to the consequent disruption of the intestinal microflora. Treatment of CDI with metronidazole and vancomycin can exacerbate the problem by continuing to disrupt the intestinal microflora. This leaves the patient susceptible to relapse or re-infection. Approximately one third of patients experience CDI relapse following treatment, and those who relapse have a greater risk of succumbing to the infection . A current imperative is the development of therapies that selectively target C. difficile, whilst leaving the intestinal microflora intact.
The C. difficile reference strain 630 encodes a single predicted sortase, CD630_27180, which has high amino-acid similarity with SrtB of S. aureus and B. anthracis. A second sortase encoded within the genome is interrupted by a stop codon prior to the catalytic cysteine and is considered a pseudogene. Thus, in contrast to other Gram-positive bacteria, C. difficile appears to have only a single functional sortase. As such, a compound that inhibits the activity of C. difficile sortase could target the pathogen without disrupting the numerous Gram-negative bacteria that make up the intestinal flora.
In this study, we demonstrate that the predicted sortase encoded by CD630_27180 recognizes and cleaves an (S/P)PXTG motif between the threonine and glycine residues. The cleavage of this motif is dependent on the conserved cysteine residue at position 209 in the predicted active site of the sortase. We have also identified seven putative sortase substrates, all of which contain the (S/P)PXTG motif. These substrates are conserved among the five C. difficile lineages and include potential adhesins, a 5' nucleotidase, and cell wall hydrolases. Furthermore, we identified a number of small-molecule inhibitors by means of an in silico screen that inhibit the activity of the C. difficile SrtB.
Conservation of the catalytically active residues of sortase
The C. difficile population structure forms at least five distinct clonal lineages that are all associated with human infection -. To determine whether SrtB is conserved between C. difficile strains, representatives for each of the five distinct clades were chosen for analysis based on the availability of a fully annotated sequence: C. difficile strains 630 for Clade 1, R20291 and CD196 (RT027) for Clade 2 , M68 and CF5 (RT017) for Clade 3 , CD305 (RT023) for Clade 4 (unpublished, WTSI), and M120 (RT078) for Clade 5 . BLAST searches of these representative strains show that srtB is conserved across all five C. difficile lineages. A second sortase-like gene in the 630 genome, classified as a pseudogene because of an in frame stop codon prior to the catalytic cysteine, is absent from the other four C. difficile lineages.
Bioinformatic prediction of sortase substrates
A bioinformatics approach was used for the preliminary identification of sortase substrate proteins in C. difficile strain 630. The predicted recognition sequence for CD630_27180 has been proposed to be (S/P)PXTG by Pallen et al., and recently to also include the sequence NVQTG, found in the surface- associated collagen binding protein CbpA, by Tulli et al.. Putative proteins were screened for the patterns (S/P)PXTG and NVQTG at the C-terminus ,. Putative candidates were then assessed for the known features of a sortase substrate: a predicted N-terminal signal peptide sequence, and a cell wall sorting signal comprising of a potential transmembrane domain following the sortase recognition sequence, and at least two consecutive basic residues (arginine or lysine) at the C-terminus -.
Identification of putative C. difficile SrtB substrates in strain 630
C-terminal sorting signal
Putative cell wall hydrolase
Putative cell surface protein, collagen binding protein
Putative membrane-associated 5'-nucleotidase/phosphoesterase
Putative cell-wall hydrolase
Putative adhesion, collagen binding protein
Collagen binding protein, CbpA
Putative surface protein
Putative cell surface protein, collagen binding protein
Purified C. difficileSrtB cleaves (S/P)PXTG peptides
FRET peptide details
Based on CD0183 sequence
Control for above peptide
Based on CD0386, CD3392 sequence
Control for above peptide
Based on CD2768 sequence
Control for above peptide
Based on CD2831 sequence
Control for above peptide
Based on CbpA sequence
Based on CD0183 sequence
Based on CD0386 sequence
Based on CD2768 sequence
Based on CD2831 sequence
Analysis of FRET reaction
Kinetic measurements of SrtB activity
Using SciPy 0.11.0 in Python 2.7.3, where V max is the apparent maximal enzymatic velocity, K m is the apparent Michaelis constant, and K i is the apparent inhibitor dissociation constant for unproductive substrate binding. This resulted in a K m of 74.7 ± 48.2 μM and a K cat of 1.1×10-3 ± 6×10-4 min-1 for d-SDSPKTGDN-e (Figure 7B). This analysis was performed for d-PVPPKTGDS-e, resulting in a K m of 53.3 ± 25.6 μM and a K cat of 8.3×10-4 ± 3×10-4 min-1. SrtBΔN26 is subject to substrate inhibition; at peptide concentrations greater than 30 μM, the rate of SrtBΔN26 activity decreases. Substrate inhibition has previously been observed for other sortase enzymes in vitro, and is not expected to be physiologically relevant .
Inhibiting SrtB activity
Structure of most effective inhibitors of SrtB ΔN26
63.1 ± 8.8 μM
60.1 ± 4.7 μM
44.1 ± 6.9 μM
C. difficile infection is invariably associated with the disruption of the normal intestinal microflora by the administration of broad spectrum antibiotics. Thus there is a pressing need to develop therapies that selectively target C. difficile while leaving the intestinal microflora intact. The C. difficile reference strain 630 encodes a single predicted sortase, CD630_27180, which has strong amino acid similarity with SrtB of S. aureus and B. anthracis. Sortase substrates frequently contribute toward pathogenesis via their involvement in attachment to specific tissues during infection ,-, as well as the bacteria's ability to evade the immune response of the host ,. Sortases, although not essential for growth or viability of the organism, are often essential for virulence in Gram-positive organisms; inactivation of sortases reduces colonization in mice ,,,, and decreases adhesion and invasion in vitro,,,,. Sortases and their substrates are considered promising targets for the development of new anti-infective compounds ,,. Unusually for Gram-positive bacteria, C. difficile appears to possess a single sortase enzyme that is likely to be important for the viability of the pathogen as we have been unable to construct a C. difficile strain 630 SrtB defined mutant (unpublished data). Inhibiting the C. difficile sortase could prove to be a strategy to specifically target C. difficile.
In this study, we cloned, expressed and characterized the sortase encoded by CD630_27180 of C. difficile 630, a predicted class B sortase (SrtB). Sortase nomenclature is based on sequence similarity to the known classes of sortase, A-F . Sortases of class B typically are involved in heme-iron uptake and tend to be expressed in operons with their substrates ,. Genes encoding class A sortases are not found in proximity to their substrates, which consist of a variety of surface proteins with diverse biological functions. Several exceptions to these rules have already been described, notably a class B sortase that polymerizes pilin subunits in S. pyogenes, and a class E sortase from C. diphtheriae that serves a housekeeping function . The potential C. difficile sortase substrates identified in this paper comprise a diverse range of surface proteins, suggesting that SrtB may serve as a housekeeping sortase in C. difficile, a function usually reserved for class A sortases.
These potential sortase substrates in C. difficile strain 630 comprise of seven proteins, all containing an (S/P)PXTG motif, that are predicted to be surface localized and are conserved across C. difficile strains. Recently it was proposed that a C. difficile collagen binding protein, CbpA, may be sorted to the cell surface by sortase recognizing an NVQTG motif . In this study, we developed a FRET-based assay to demonstrate that SrtB of C. difficile recognizes and cleaves the (S/P)PXTG motif between the threonine and glycine residues, and that cleavage is dependent on a single cysteine residue at position 209. SrtBΔN26 does not appear to cleave the S. aureus SrtA and SrtB motifs, LPXTG and NPQTN, respectively, nor the NVQTG motif in vitro, suggesting that CbpA from C. difficile may be attached to the cell surface by another mechanism.
The FRET-based assay enabled us to determine kinetic parameters for the recombinant C. difficile SrtB. Although the catalytic activity appears low, low catalytic efficiency is observed for most sortases in vitro,. The kinetic and cleavage data we report for SrtBΔN26 is consistent with this trend. In vivo, the sorting motifs are part of a larger protein, and the transpeptidation substrates are part of a cell wall precursor or mature peptidoglycan ,,. The transpeptidation reaction has been observed in vitro for sortases from bacteria with a Lys-type peptidoglycan, where cross-linking occurs through a peptide bridge , such as S. aureus and Streptococcus species ,,, but not for bacteria with Dap-type peptidoglycan such as Bacillus with direct cross-linkages through m-diaminopimelic acid . The likely cell wall anchor of the C. difficile SrtB substrates is the diaminopimelic acid cross-link , similar to Bacillus. When transpeptidation is observed in vitro, the cleavage efficiency of sortase increases.
This study revealed that recombinant SrtBΔN26 cleaves the (S/P)PXTG motifs with varying levels of efficiency, cleaving the sequences PPKTG and SPQTG with the greatest efficiency. Apparent preferential cleavage efficiency of certain substrate sequences in vitro has been observed in other sortases. For example, in B. anthracis, BaSrtA cleaves LPXTG peptides more readily than a peptide containing the sequence LPNTA . The biological significance of this peptide sequence preference is unknown.
Small-molecule inhibitors with activity against SrtA and SrtB have been reported that prevent cleavage of fluorescently-labelled peptide compounds by sortase in vitro. These compounds inhibit cell adhesion to fibronectin, yet, they have no effect on in vitro growth. Inhibitors tested against SrtA, SrtB and SrtC in B. anthracis irreversibly modified the active cysteine residue . Several compounds identified in this study had an inhibitory effect on C. difficile SrtB activity. However, these lead compounds had no direct effect on in vitro C. difficile growth (data not shown), which is consistent with observations in S. aureus. Inhibition of bacterial growth is not considered vital in the development of sortase-based drug therapies. In both Staphylococcus and Bacillus, sortase inhibitors show good suitability for further development as therapeutics despite their lack of bactericidal activity. When mice challenged with S. aureus were treated with sortase inhibitor compounds, infection rates and mortality were reduced , despite these compounds having no effect on staphylococcal growth . The use of in silico approaches such as the LeadBuilder method employed by this study to screen databases of putative small-molecule inhibitors for further analysis has been validated. Further analysis of the structural similarities between the hit compounds could lead to a refinement of SrtB inhibitor design and increased potency in vitro.
In conclusion, we demonstrate that C. difficile encodes a single sortase, SrtB, with in vitro activity. We have confirmed the C. difficile SrtB recognition sequence as (S/P)PXTG, and show that C. difficile SrtB cleaves the (S/P)PXTG motif within peptides between the threonine and glycine residues. The cysteine residue within the predicted active site is essential for activity of the enzyme, and the cleavage of fluorescently-labelled peptides can be inhibited by MTSET, a known cysteine protease inhibitor. SrtB inhibitors identified through our in silico screen show a greater level of efficacy then MTSET at inhibiting the protease activity of C. difficile SrtB. Such inhibitors provide a significant step in successfully identifying C. difficile SrtB inhibitor compounds, which can be further refined to enhance their efficacy, and may contribute towards the development of novel selective therapeutics against CDI.
C. difficile strain 630  was cultured on Brazier’s agar (BioConnections) supplemented with 4% egg yolk (BioConnections) and 1% defibrinated horse blood (TCS Biosciences Ltd.). Liquid cultures were grown in brain heart infusion broth (Oxoid Ltd.) supplemented with 0.05% L-cysteine (BHIS broth). All media was supplemented with C. difficile antibiotic supplement (250 μg/ml D-cycloserine and 8 μg/ml cefoxitin, BioConnections). C. difficile cultures were incubated at 37°C for 24-48 hours in a Whitley MG500 anaerobic workstation (Don Whitley Scientific Ltd.).
One Shot Top10® (Invitrogen) and XL-1 Blue (Agilent) Escherichia coli were used for all cloning steps, and NiCo21(DE3) E. coli (NEB) was used for the expression of recombinant proteins . E. coli strains were grown at 37°C on Luria-Bertani (LB) agar (Novagen) or in LB broth (Difco). Media was supplemented with 100 μg/ml ampicillin or 50 μg/ml kanamycin as required.
Genomic DNA isolation
Genomic DNA was isolated from C. difficile strain 630 , by phenol chloroform extraction as previously described  and used as a template for cloning. The annotated genome sequences from C. difficile strains R20291 and CD196 (RT027) , M68 and CF5 (RT017) , M120 (RT078) , and CD305 (RT023) (unpublished, Wellcome Trust Sanger Institute) were used for analysis.
Identification of sortase substrates
All proteins encoded by C. difficile strain 630 , were searched for the patterns (S/P)PXTG  and NVQTG  positioned 17-45 amino acid residues from the C-terminus . The resulting candidate protein list was assessed for the known features of a sortase substrate: (i) a suitable N-terminal signal peptide sequence, as predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) , (ii) a potential transmembrane segment following the C-terminal "LPXTG-like" sequence, as predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) , and (iii) at least two consecutive basic residues (arginine or lysine) at the C-terminus -.
Primers used in this study
Plasmids used in this study
Codon optimized srtB, synthesized and cloned in pQE30xa
Obtained from Celtek Bioscience, LLC
Commercial protein expression vector
Provided by Neil Fairweather
Codon optimized srtB cloned in pET28a
pET28a- srtB ΔN26
srtB with residues 2-25 replaced with a six-histidine tag
pET28a- srtB ΔN26,C209A
Same as above, with C209A substitution
Total RNA was isolated from C. difficile 630 grown in BHIS at early exponential phase (t = 3 hours, OD600 = 0.4-0.5), late exponential phase (t = 5'hours, OD600 = 0.8-0.9), and stationary phase (t = 24 hours, OD600 = 0.6-0.8) using RNAprotect Bacteria Reagent (Qiagen) and the FastRNA Pro Blue Kit (MP Biomedicals LLC., Illkirch, France) in accordance with the manufacturer’s instructions. Genomic DNA was removed from total RNA samples using TURBO DNase (Life Technologies). Equal amounts of RNA were reverse transcribed into complementary DNA (cDNA) for expression analysis. Briefly, one μg random primers (Invitrogen) and 40 units RNasin Plus RNase inhibitor (Promega) was added to one μg RNA in a final volume of 10 μl, and incubated at 65'C for 10 min followed by room temperature for 30 min. The following first-strand mixture was added for cDNA synthesis: four μl of 5x first-strand buffer (Invitrogen), two μl 0.1 M DTT (Invitrogen), two μl 10 mM dNTP mix (New England BioLabs), and 1.5'μl Superscript II (Invitrogen). The reaction mixture was incubated at 25'C for 10 minutes, 42°C for 1 h, and finally 70°C for 15'minutes. RT-PCRs were performed with gene specific primers (Additional file 2: Table S1) using cDNA as a template.
Purification of recombinant protein
Expression constructs were transformed into E. coli NiCo21(DE3) (NEB). Cultures grown at 37°C were induced for expression with 1 mM IPTG when the OD600 reached 0.6, and harvested after 5'hours. Cell pellets were resuspended in lysis buffer [1× Bugbuster (Novagen), 50 mM NaH2PO4, 300 mM NaCl, 40 mM imidazole, 1 mM DTT, 1 mg/ml lysozyme, and 25 U/ml Benzonase nuclease (Novagen) (pH 7.5)]. Lysates were sonicated on ice for 2 min (15'sec on/off) at 50% Vibra Cell™ high intensity ultrasonic processor (Jencon, Leighton Buzzard, Bedfordshire, UK) before centrifugation at 10,000 rpm for 45'min. The supernatant was passed through a 0.22 μM filter before applying to a 1 ml HisTrap HP column (GE Healthcare), pre-equilibrated with buffer (50 mM NaH2PO4, 300 mM NaCl, 40 mM imidazole, 1 mM DTT, pH 7.5). SrtBΔN26 was eluted with an imidazole gradient (40 - 500 mM) over 25 column volumes. Fractions containing SrtBΔN26 (as identified by SDS-PAGE) were pooled and injected onto a HiLoad 16/60 Superdex 200 column (GE Healthcare) pre-equilibrated with buffer F (5'mM CaCl2, 50 mM Tris hCl (pH 7.5), 150 mM NaCl, 1 mM DTT). Eluted fractions containing SrtBΔN26 were pooled and concentrated using an Amicon Ultra-15 (10 kDa) centrifuge filter unit (Millipore). Protein samples were quantified using Bradford reagent (Thermo Scientific) and analyzed by SDS-PAGE. The mutant protein SrtBΔN26,C209A was expressed and purified following the above method. Expression of SrtBΔN26 and SrtBΔN26,C209A was confirmed by MALDI fingerprinting.
Samples were resolved on Novex NuPage 10% Bis-Tris SDS-PAGE gels (Invitrogen) before transferring to Hybond-C Extra nitrocellulose (GE Healthcare). Membranes were probed with rabbit antiserum directed against 6xHis-tag (1:5000, Abcam), followed by goat anti-rabbit IRDye conjugated secondary antibody (1:7500, LI-COR Biotechnology). Blots were visualized using an Odyssey near-infrared imager (LI-COR Biotechnology).
In vitroanalysis of sortase activity
SrtBΔN26 activity was monitored using a fluorescence resonance energy transfer (FRET) assay  in buffer F (5'mM CaCl2, 50 mM Tris hCl (pH 7.5), 150 mM NaCl, and 1 mM DTT). Fluorescently self-quenched peptides tagged with 5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid (Edans) as a fluorophore and 4-([4-(Dimethylamino)phenyl]azo)-benzoic acid (Dabcyl) as a quencher , and containing the predicted sorting signals of SrtB were purchased from Protein Peptide Research Ltd and solubilized in DMSO (Table 2). The FRET-based assay was performed in a final volume of 100 μl buffer F containing 10 μM SrtBΔN26 and 20 μM fluorogenic peptide in clear-bottomed, black polystyrene 384-well plates (Nunc). Plates were incubated for 48 hours at 37°C, during which fluorescence (excitation = 340 nm, emission = 490 nm) was measured using a SpectraMax M3 plate reader (Molecular Devices). Five mM 2-(trimethylamonium)ethylmethanethiosulfonate (MTSET, Affymetrix) was added to the reaction as indicated. Each experiment was performed in triplicate with a minimum of three biological replicates, and the results are presented as the means and the standard error of the data obtained. The two-tailed Student’s T-test was used to analyze the data. MALDI analysis of FRET reaction samples was performed by the Protein and Nucleic Acid Chemistry Facility (University of Cambridge) to determine exact cleavage site within each peptide.
Kinetic data for SrtBΔN26 were obtained by incubating varying concentrations of peptide (8, 10, 20, 40, 80, 160, 200 and 240 μM) with 10 μM SrtBΔN26. All reactions were performed as described above, with fluorescence monitored every ten minutes over a 13 hour period. To correlate fluorescence signal, expressed as arbitrary relative fluorescence units (RFU), with the concentration of product formed, standard curves of the fluorophore Edans were collected. The linear segment of the fluorophore standard curve generated a conversion ratio of 703.9 RFU/ μM Edans. Initial velocities (V) were determined from the progress curves and plotted against substrate concentration [S]. The data were fitted to a modified version of the Michaelis-Menten equation incorporating substrate inhibition using SciPy 0.11.0 in Python 2.7.3, where V max is the maximal enzymatic velocity, K m is the Michaelis constant, and K i is the inhibitor dissociation constant for unproductive substrate binding. All data points were collected in triplicate, and the overall assay was run in duplicate.
Identification of SrtB inhibitors
The proprietary LeadBuilder virtual screening method (Domainex, Ltd) was used to interrogate a database (PROTOCATS) of 80,000 potential compounds which had been pre-selected as protease inhibitors. The virtual screening protocol used pharmacophoric and docking filters derived from analysis of the BaSrtB crystal structure (with which the C. difficile SrtB shows 70% identity and 90% similarity at the active site). Sixty-two compounds identified in this screen as potential SrtB inhibitors were obtained from Enamine, ChemBridge, and Key Organics, and solubilized in DMSO. Selected compounds and MTSET were incubated with 10 μM SrtBΔN26 at a range of concentrations in the FRET-based assay conditions described above, so that final DMSO concentrations were ≤ 3.75%, a concentration shown to have no significant effect on control fluorescence (data not shown). IC50 values were determined by non-linear least squares fit to a four parameter sigmoidal function using SciPy 0.11.0 in Python 2.7.3.
Fluorescence resonance energy transfer
- Dabcyl or d :
- Edans or e :
C. difficile infection
Relative fluorescence units
We thank Jun Wheeler for MALDI mass spectrometry fingerprinting analysis of recombinant proteins; Mark Donahue for assistance with data analysis; Hayley Angove and Wendy Savory for assistance with development of the FRET-based assay and sortase protein expression, respectively. We thank Neil Fairweather, Johann Peltier, Helen A. Shaw and Madeleine Moule for critical reading of the manuscript.
This research was supported by funding from Wellcome Trust grant number 086418/Z/ and MRC grant number 499 94717.
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