Structural and biochemical characterizations of the novel autolysin Acd24020 from Clostridioides difficile and its full-function catalytic domain as a lytic enzyme.

Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding (CWB) domains, to be involved in different physiological functions that require bacterial cell wall remodeling. We identified a novel autolysin, Acd24020, from Clostridioides (Clostridium) difficile (C. difficile), with an endopeptidase catalytic domain belonging to the NlpC/P60 family and three bacterial Src-homology 3 domains as CWB domains. The catalytic domain of Acd24020 (Acd24020-CD) exhibited C. difficile-specific lytic activity equivalent to Acd24020, indicating that Acd24020-CD has full-function as a lytic enzyme by itself. To elucidate the specific peptidoglycan-recognition and catalytic reaction mechanisms of Acd24020-CD, biochemical characterization, X-ray structure determination, a modeling study of the enzyme/substrate complex, and mutagenesis analysis were performed. Acd24020-CD has an hourglass-shaped substrate-binding groove across the molecule, which is responsible for recognizing the direct 3-4 cross-linking structure unique to C. difficile peptidoglycan. Based on the X-ray structure and modeling study, we propose a dynamic Cys/His catalyzing mechanism, in which the catalytic Cys299 and His354 residues dynamically change their conformations to complement each step of the enzyme catalytic reaction.

The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli.

Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores.IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.

The C-Terminal Domain of Clostridioides difficile TcdC Is Exposed on the Bacterial Cell Surface.

Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (OB-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors.IMPORTANCE The transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an anti-sigma factor requires a cytoplasmic localization of the C terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiTopt) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development.

The Structure of Clostridioides difficile SecA2 ATPase Exposes Regions Responsible for Differential Target Recognition of the SecA1 and SecA2-Dependent Systems.

SecA protein is a major component of the general bacterial secretory system. It is an ATPase that couples nucleotide hydrolysis to protein translocation. In some Gram-positive pathogens, a second paralogue, SecA2, exports a different set of substrates, usually virulence factors. To identify SecA2 features different from SecA(1)s, we determined the crystal structure of SecA2 from Clostridioides difficile, an important nosocomial pathogen, in apo and ATP-γ-S-bound form. The structure reveals a closed monomer lacking the C-terminal tail (CTT) with an otherwise similar multidomain organization to its SecA(1) homologues and conserved binding of ATP-γ-S. The average in vitro ATPase activity rate of C. difficile SecA2 was 2.6 ± 0.1 µmolPi/min/µmol. Template-based modeling combined with evolutionary conservation analysis supports a model where C. difficile SecA2 in open conformation binds the target protein, ensures its movement through the SecY channel, and enables dimerization through PPXD/HWD cross-interaction of monomers during the process. Both approaches exposed regions with differences between SecA(1) and SecA2 homologues, which are in agreement with the unique adaptation of SecA2 proteins for a specific type of substrate, a role that can be addressed in further studies.

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Lead Optimization Yields High Affinity Frizzled 7-Targeting Peptides That Modulate Clostridium difficile Toxin B Pathogenicity in Epithelial Cells.

Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.

Are Clostridium difficile toxins nephrotoxic?

Clostridium difficile-associated disease (CDAD) occurs along a spectrum from simple uncomplicated enteritis to a multi-system disease which may include nephropathy. Pathology is attributed to bacterial toxins, but it is unclear if the latter are directly nephrotoxic. Anecdotes of renal disease from human biopsy findings suggest a variation of histopathologies, but data are relatively limited. Acute renal failure does occur in patients with advanced morbidity. CDAD can complicate chronic renal failure. Kidney tissue culture cytotoxicity has long been known. Kidney function alterations among animal models or diseased humans are relatively uncommon in mild to moderate enteritis. Rare findings of toxinemia are reported. Some have proposed that renal dysfunction arises more from pre-renal compromises. Direct toxin studies on whole kidney are sparse. The role of direct toxin-associated renal disease is worthy of further investigation given the current impetus towards the development of protective and therapeutic passive and active immunity. Hypotheses of toxin-direct or pre-renal toxin compromise of renal function prevail.

The Antimicrobial Peptide CopA3 Inhibits Clostridium difficile Toxin A-Induced Viability Loss and Apoptosis in Neural Cells.

Numerous studies have reported that enteric neurons involved in controlling neurotransmitter secretion and motility in the gut critically contribute to the progression of gut inflammation. Clostridium difficile toxins, which cause severe colonic inflammation, are also known to affect enteric neurons. Our previous study showed that C. difficile toxin A directly induces neural cell toxicities, such as viability loss and apoptosis. In the current study, we attempted to identify a potent inhibitor of toxin A-induced neural cell toxicity that may aid in managing toxin A-induced gut inflammation. In our recent study, we found that the Korea dung beetle-derived antimicrobial peptide CopA3 completely blocked neural cell apoptosis caused by okadaic acid or 6-OHDA. Here, we examined whether the antimicrobial peptide CopA3 inhibited toxin A-induced neural cell damage. In neuroblastoma SH-SY5Y cells, CopA3 treatment protected against both apoptosis and viability loss caused by toxin A. CopA3 also completely inhibited activation of the pro-apoptotic factor, caspase-3. Additionally, CopA3 rescued toxin A-induced downregulation of neural cell proliferation. However, CopA3 had no effect on signaling through ROS/p38 MAPK/p27kip1, suggesting that CopA3 inhibits toxin A-induced neural cell toxicity independent of this well-characterized toxin A pathway. Our data further suggest that ability of CopA3 to rescue toxin A-induced neural cell damage may also ameliorate the gut inflammation caused by toxin A.

Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis.

Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB), that damage the colonic epithelial barrier and induce inflammatory responses. The function of the colonic vascular barrier during CDI has been relatively understudied. Here we report increased colonic vascular permeability in CDI mice and elevated vascular endothelial growth factor A (VEGF-A), which was induced in vivo by infection with TcdA- and/or TcdB-producing C. difficile strains but not with a TcdA-TcdB- isogenic mutant. TcdA or TcdB also induced the expression of VEGF-A in human colonic mucosal biopsies. Hypoxia-inducible factor signalling appeared to mediate toxin-induced VEGF production in colonocytes, which can further stimulate human intestinal microvascular endothelial cells. Both neutralization of VEGF-A and inhibition of its signalling pathway attenuated CDI in vivo. Compared to healthy controls, CDI patients had significantly higher serum VEGF-A that subsequently decreased after treatment. Our findings indicate critical roles for toxin-induced VEGF-A and colonic vascular permeability in CDI pathogenesis and may also point to the pathophysiological significance of the gut vascular barrier in response to virulence factors of enteric pathogens. As an alternative to pathogen-targeted therapy, this study may enable new host-directed therapeutic approaches for severe, refractory CDI.

Clostridium difficile colonization among patients with clinically significant diarrhea and no identifiable cause of diarrhea.

OBJECTIVE: To determine the prevalence of Clostridium difficile colonization among patients who meet the 2017 IDSA/SHEA C. difficile infection (CDI) Clinical Guideline Update criteria for the preferred patient population for C. difficile testing. DESIGN: Retrospective cohort. SETTING: Tertiary-care hospital in St. Louis, Missouri.PatientsPatients whose diarrheal stool samples were submitted to the hospital's clinical microbiology laboratory for C. difficile testing (toxin EIA) from August 2014 to September 2016.InterventionsElectronic and manual chart review were used to determine whether patients tested for C. difficile toxin had clinically significant diarrhea and/or any alternate cause for diarrhea. Toxigenic C. difficile culture was performed on all stool specimens from patients with clinically significant diarrhea and no known alternate cause for their diarrhea. RESULTS: A total of 8,931 patients with stool specimens submitted were evaluated: 570 stool specimens were EIA positive (+) and 8,361 stool specimens were EIA negative (-). Among the EIA+stool specimens, 107 (19% of total) were deemed eligible for culture. Among the EIA- stool specimens, 515 (6%) were eligible for culture. One EIA+stool specimen (1%) was toxigenic culture negative. Among the EIA- stool specimens that underwent culture, toxigenic C. difficile was isolated from 63 (12%). CONCLUSIONS: Most patients tested for C. difficile do not have clinically significant diarrhea and/or potential alternate causes for diarrhea. The prevalence of toxigenic C. difficile colonization among EIA- patients who met the IDSA/SHEA CDI guideline criteria for preferred patient population for C. difficile testing was 12%.

Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis.

Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis.

Time-kill kinetics of cadazolid and comparator antibacterial agents against different ribotypes of Clostridium difficile.

PURPOSE: Clostridium difficile infection (CDI) is an increasing cause of nosocomial diarrhoea worldwide, which has been partly attributed to the emergence of hypervirulent strains including C. difficile BI/NAP1/ribotype 027 and BK/NAP7/ribotype 078. Cadazolid is a new antibiotic currently in late-stage clinical studies for the treatment of CDI. The present study evaluated the in vitro bactericidal effect of cadazolid and comparator antibiotics against four C. difficile strains. The data demonstrate the potent and bactericidal activity of cadazolid against different ribotypes of C. difficile. METHODOLOGY: MICs for test antibiotics were determined in brain- heart infusion-supplemented broth (BHIS) containing 5 g l-1 yeast extract and 0.025 % (w/v) l-cysteine. Time-kill kinetics to investigate the rate of killing of each antibiotic at sub- and supra-MIC concentrations were performed at concentrations of 0.5, 1, 2, 4, 8 or 16× the MIC of cadazolid, vancomycin and fidaxomicin at intervals over a 48 h period.Results/key findings. Cadazolid-mediated killing of C. difficile was faster and occurred at lower concentrations than observed for vancomycin, while potency and killing was largely comparable to those observed for fidaxomicin. Notably, cadazolid also displayed a potent bactericidal effect against fluoroquinolone-resistant hypervirulent ribotype 027 and 078 strains. C. difficile spore formation was largely inhibited by all three antibiotics at concentrations >1× MIC; however, none were able to eliminate spores effectively, which were present at the start of the experiment. CONCLUSION: The data presented here demonstrate the potent in vitro bactericidal activity of cadazolid against different ribotypes of C. difficile, although on a limited set of strains.

Toxigenic Clostridium difficile isolates from clinically significant diarrhoea in patients from a tertiary care centre.

BACKGROUND & OBJECTIVES: Clostridium difficile is the primary cause of hospital-acquired colitis in patients receiving antibiotics. The pathogenicity of the organism is mainly due to the production of toxins. This study was conducted to investigate the presence of toxigenic C. difficile in the faecal samples of hospitalized patients suspected to have C. difficile infection (CDI) and corroborating the findings with their clinical and demographic data. METHODS: Diarrhoeic samples obtained from 1110 hospitalized patients were cultured for C. difficile and the isolates confirmed by phenotypic and molecular methods. Toxigenicity of the isolates was determined using enzyme-linked immunosorbent assay for toxins A and B. Details of patients included in the study were noted and analyzed. RESULTS: Of the 1110 patients (mean age 39±19.6 yr), 63.9 per cent were males and 36.1 per cent were females. The major antibiotics received by the patients were nitazoxanide (23.9%), penicillins/penicillin combinations (19.0%), quinolones including fluoroquinolones (13.1%), carbapenems (11.5%), glycopeptides (11.0%) and cephalosporins (8.4%). The clinical symptoms predominantly present were watery diarrhoea (56.4%), fever (40.0%) and abdominal pain (35.3%). The underlying diseases were gastrointestinal disorders (52.6%), followed by cancers (13.2%), surgical conditions (8.3%), and hepatic disorders (8.0%). Of the 174 C. difficile isolates, 54.6 per cent were toxigenic. Toxigenic C. difficile was present in all patients with surgical conditions, 65.2 per cent with cancers and 57.1 per cent with gastrointestinal disorders. INTERPRETATION & CONCLUSIONS: C. difficile was found to be an important cause of gastrointestinal infections in hospitalized patients with underlying diseases and on antibiotics. Clinical conditions of the patients correlating with toxigenic culture can be an important tool for establishing CDI diagnosis.

Structural and functional insights into corrinoid iron-sulfur protein from human pathogen Clostridium difficile.

The human pathogen Clostridium difficile infection (CDI) is one of the most important healthcare-associated infections. The Wood-Ljungdahl pathway, which is responsible for Acetyl-CoA biosynthesis, is essential for the survival of the pathogen and is absent in humans. The key proteins and enzymes involved in the pathway are attractive targets for the treatment of CDI. Corrinoid iron-sulfur protein (CoFeSP) is a key protein and acts as a methyl transformer in the Wood-Ljungdahl pathway. In this study, CoFeSP from Clostridium difficile (CoFeSPCd) was cloned, expressed in E. coli and characterized for the first time. The structure and function of CoFeSPCd were investigated using homology structure modeling, spectroscopy, electrochemistry, steady state/pre-steady state kinetics and molecular docking. The two metal centers of CoFeSPCd, corrinoid cofactor and [4Fe-4S] cluster, were characterized using metal analysis, structural modeling, UV-Vis, EPR and direct electrochemistry. The methyl transfer activity between CH3-H4folate (CH3-THF) and CoFeSPCd catalyzed by methyl transferase (MeTrCd) was determined by kinetic studies. These results provide a molecular basis for innovative drug design and development to treat human CDI.

Positive predictive value of the immunoassay for Clostridium difficile toxin A and B detection at a private hospital. / Valor predictivo positivo de la prueba de inmunoanálisis para detección de toxina A y B de Clostridium difficile en un hospital privado.

INTRODUCTION: Clostridium difficile (C. difficile) is a Gram-positive bacillus that is a common cause of diarrhea in the hospital environment, with a documented incidence of up to 10%. There are different methods to detect it, but a widely used test in our environment is the immunoassay for toxins A and B. AIMS: The aim of our study was to 1) estimate the positive predictive value of the immunoassay for the detection of the C. difficile toxins A and B, 2) to establish the incidence of C. difficile-associated diarrhea in the hospital, and 3) to know the most common associated factors. METHODS: A diagnostic test accuracy study was conducted within the time frame of January 2010 to August 2013 at the Hospital Christus Muguerza® Alta Especialidad on patients with symptoms suggestive of C. difficile-associated diarrhea that had a positive immunoassay test and confirmation of C. difficile through colon biopsy and stool culture. RESULTS: The immunoassay for toxins A and B was performed in 360 patients. Fifty-five of the cases had positive results, 35 of which showed the presence of C. difficile. Incidence was 10.2% and the positive predictive value of the test for C. difficile toxins A and B was 0.64 (95% CI, 0.51-0.76). Previous antibiotic therapy (n=29) and proton pump inhibitor use (n=19) were the most common associated factors. CONCLUSIONS: C. difficile incidence in our environment is similar to that found in the literature reviewed, but the positive predictive value of the test for toxin A and B detection was low.

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix.

Structural Basis of Proline-Proline Peptide Bond Specificity of the Metalloprotease Zmp1 Implicated in Motility of Clostridium difficile.

Clostridium difficile is a pathogenic bacterium causing gastrointestinal diseases from mild diarrhea to toxic megacolon. In common with other pathogenic bacteria, C. difficile secretes proteins involved in adhesion, colonization, and dissemination. The recently identified Zmp1 is an extracellular metalloprotease showing a unique specificity for Pro-Pro peptide bonds. The endogenous substrates of Zmp1 are two surface proteins implicated in adhesion of C. difficile to surface proteins of human cells. Thus, Zmp1 is believed to be involved in the regulation of the adhesion-motility balance of C. difficile. Here, we report crystal structures of Zmp1 from C. difficile in its unbound and peptide-bound forms. The structure analysis revealed a fold similar to Bacillus anthracis lethal factor. Crystal structures in the open and closed conformation of the S-loop shed light on the mode of binding of the substrate, and reveal important residues for substrate recognition and the strict specificity of Zmp1 for Pro-Pro peptide bonds.

Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity.

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.

Structure and function of a Clostridium difficile sortase enzyme.

Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. In some gram-positive bacteria (e.g. Staphylococcus aureus), sortases have been found to be essential for pathogenesis and their inhibitors are under development as potential novel therapeutics. Here we provide the first report on the structural characterisation of the C. difficile sortase. An active site mutant was crystallised and its structure determined to 2.55 Å by X-ray diffraction to provide structural insight into its catalytic mechanism. In order to elucidate the role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB and that an SPKTG peptide motif is involved in the transpeptidation reaction with the C. difficile peptidoglycan. In an animal model for C. difficile infection, the SrtB mutant caused disease at a similar rate of onset as the wild type strain. In conclusion, our detailed study shows that the SrtB enzyme from C. difficile does not play an essential role in pathogenesis.