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Significance Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile infection (CDI) are increasing, making C. difficile an important public health concern. As a toxin-mediated disease, there is significant interest in understanding the receptors that mediate the cellular entry and function of these toxins. The targeted disruption of toxin-receptor interactions could provide novel therapeutic strategies that can either augment or replace the need for antibiotic therapies in the treatment of CDI. 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.

A channel for the transport of hydrosulphide ions in Clostridium difficile is identified and shown to be polyspecific, being a member of the formate/nitrite transporter family. Keeping hydrogen sulphide at bay Hydrogen sulphide (H 2 S) and the hydrosulphide ion (HS − ) are metabolic products of anaerobic bacterial growth, and are thought to have had a crucial role in the emergence of life on Earth. At high concentrations, both are toxic to the cell, and therefore call for a release mechanism. For many years, such a channel has remained elusive, but now Bryan Czyzewski and Da-Neng Wang have used genetic, biochemical and structural approaches to identify and characterize a hydrosulphide ion channel in the pathogen Clostridium difficile . The channel is a member of the formate/nitrite transport family, and is polyspecific, facilitating the transport of HS − , formate and nitrite. Its low open probability is consistent with its physiological role in the cell. The hydrosulphide ion (HS − ) and its undissociated form, hydrogen sulphide (H 2 S), which are believed to have been critical to the origin of life on Earth 1 , remain important in physiology and cellular signalling 2 . As a major metabolite in anaerobic bacterial growth, hydrogen sulphide is a product of both assimilatory and dissimilatory sulphate reduction 2 , 3 , 4 . These pathways can reduce various oxidized sulphur compounds including sulphate, sulphite and thiosulphate. The dissimilatory sulphate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, in which process it produces excess amounts of H 2 S (ref. 4 ). The reduction of sulphite is a key intermediate step in all sulphate reduction pathways. In Clostridium and Salmonella , an inducible sulphite reductase is directly linked to the regeneration of NAD + , which has been suggested to have a role in energy production and growth, as well as in the detoxification of sulphite 3 . Above a certain concentration threshold, both H 2 S and HS − inhibit cell growth by binding the metal centres of enzymes and cytochrome oxidase 5 , necessitating a release mechanism for the export of this toxic metabolite from the cell 5 , 6 , 7 , 8 , 9 . Here we report the identification of a hydrosulphide ion channel in the pathogen Clostridium difficile through a combination of genetic, biochemical and functional approaches. The HS − channel is a member of the formate/nitrite transport family, in which about 50 hydrosulphide ion channels form a third subfamily alongside those for formate 10 , 11 (FocA) and for nitrite 12 (NirC). The hydrosulphide ion channel is permeable to formate and nitrite as well as to HS − ions. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the channel’s crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.

Clostridium difficile toxinotypes are groups of strains defined by changes in the PaLoc region encoding two main virulence factors: toxins TcdA and TcdB. Currently, 24 variant toxinotypes (I-XXIV) are known, in addition to toxinotype 0 strains, which contain a PaLoc identical to the reference strain VPI 10463. Variant toxinotypes can also differ from toxinotype 0 strains in their toxin production pattern. The most-studied variant strains are TcdA-, TcdB+ (A-B+) strains and binary toxin CDT-producing strains. Variations in toxin genes are also conserved on the protein level and variant toxins can differ in size, antibody reactivity, pattern of intracellular targets (small GTPases) and consequently in their effects on the cell. Toxinotypes do not correlate with particular forms of disease or patient populations, but some toxinotypes (IIIb and VIII) are currently associated with disease of increased severity and outbreaks worldwide. Variant toxinotypes are very common in animal hosts and can represent from 40% to 100% of all isolates. Among human isolates, variant toxinotypes usually represent up to 10% of strains but their prevalence is increasing.

Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The two primary toxins, TcdA and TcdB, disrupt host cell function by inactivating small GTPases that regulate the actin cytoskeleton. This review will discuss the role of these two toxins in pathogenesis and the structural and molecular mechanisms by which they intoxicate cells. A focus will be placed on recent publications highlighting mechanistic similarities and differences between TcdA, TcdB, and different TcdB variants.

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 toxins TcdA and TcdB enhance pathogenesis by inducing vascular endothelial growth factor A (VEGF-A) production and promoting colonic vascular permeability.

Chlorhexidine is a broad-spectrum antimicrobial commonly used to disinfect the skin of patients to reduce the risk of healthcare-associated infections. Because chlorhexidine is not sporicidal, it is not anticipated that it would have an impact on skin contamination with Clostridium difficile, the most important cause of healthcare-associated diarrhea. However, although chlorhexidine is not sporicidal as it is used in healthcare settings, it has been reported to kill spores of Bacillus species under altered physical and chemical conditions that disrupt the spore's protective barriers (e.g., heat, ultrasonication, alcohol, or elevated pH). Here, we tested the hypothesis that similarly altered physical and chemical conditions result in enhanced sporicidal activity of chlorhexidine against C. difficile spores. C. difficile spores became susceptible to heat killing at 80 °C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04%-0.0004% w/v chlorhexidine solutions at 80 °C. Reduction of spores exposed to 4% w/v chlorhexidine solutions at moderate temperatures (37 °C and 55 °C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55 °C. Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions. Physical and chemical conditions that alter the protective barriers of C. difficile spores convey sporicidal activity to chlorhexidine. Further studies are necessary to identify additional agents that may allow chlorhexidine to reach its target within the spore.

Clostridium difficile is a Gram-positive bacillus and is the leading cause of toxin-mediated nosocomial diarrhea following antibiotic use. C. difficile flagella play a role in colonization, adherence, biofilm formation, and toxin production, which might contribute to the overall virulence of certain strains. Human and animal studies indicate that anti-flagella immune responses may play a role in protection against colonization by C. difficile and subsequent disease outcome. Here we report that recombinant C. difficile flagellin (FliC) is immunogenic and protective in a murine model of C. difficile infection (CDI) against a clinical C. difficile strain, UK1. Passive protection experiments using anti-FliC polyclonal serum in mice suggest this protection to be antibody-mediated. FliC immunization also was able to afford partial protection against CDI and death in hamsters following challenge with C. difficile 630Δerm. Additionally, immunization against FliC does not have an adverse effect on the normal gut flora of vaccinated hamsters as evidenced by comparing the fecal microbiome of vaccinated and control hamsters. Therefore, the use of FliC as a vaccine candidate against CDI warrants further testing.

Background. We evaluated the frequency of recovery of pathogens from children with diarrhea who presented to a pediatric emergency department and characterized the associated illnesses, to develop guidelines for performing a bacterial enteric culture. Methods. We conducted a prospective cohort study of all patients with diarrhea who presented to a large regional pediatric emergency department during the period from November 1998 through October 2001. A thorough microbiologic evaluation was performed on stool specimens, and the findings were correlated with case, physician, and laboratory data. Results. A total of 1626 stool specimens were studied to detect diarrheagenic bacteria and, if there was a sufficient amount of stool, Clostridium difficile toxin (688 specimens), parasites (656 specimens), and viruses (417 specimens). One hundred seventy-six (47%) of 372 specimens that underwent complete testing yielded a bacterial pathogen (Shiga toxin-producing Escherichia coli, 39 specimens [of which 28 were serotype O157:H7]; Salmonella species, 39; Campylobacter species, 25; Shigella species, 14; and Yersinia enterocolitica, 2), a viral pathogen (rotavirus, 85 specimens; astrovirus, 27; adenovirus, 18; or rotavirus and astrovirus, 8), a diarrheagenic parasite (5 specimens); or C. difficile toxin (46 specimens). Samples from 2 patients yielded both bacterial and viral pathogens. A model to identify predictors of bacterial infection found that international travel, fever, and the passing of >10 stools in the prior 24 h were associated with the presence of a bacterial pathogen. Physician judgment regarding the need to perform a stool culture was almost as accurate as the model in predicting bacterial pathogens. Conclusions. Nearly one-half of the patients who presented to the emergency department with diarrhea had a definite or plausible pathogen in their stool specimens. We were unable to develop a model that was substantially better than physician judgment in identifying patients for whom bacterial culture would yield positive results. The unexpectedly high rate of C. difficile toxin warrants further examination.

Clostridium difficile TcdA is a large toxin that binds carbohydrates on intestinal epithelial cells. A 2-Å resolution cocrystal structure reveals two molecules of α-Gal-(1,3)-β-Gal-(1,4)-β-GlcNAcO(CH 2 ) 8 CO 2 CH 3 binding in an extended conformation to TcdA. Residues forming key contacts with the trisaccharides are conserved in all seven putative binding sites in TcdA, suggesting a mode of multivalent binding that may be exploited for the rational design of novel therapeutics.