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Abstract

C lostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C . difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C . difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete.

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Most cited references 41

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Bacterial adhesion and entry into host cells.

Javier Pizarro-Cerdá, Pascale Cossart (2006)

Successful establishment of infection by bacterial pathogens requires adhesion to host cells, colonization of tissues, and in certain cases, cellular invasion-followed by intracellular multiplication, dissemination to other tissues, or persistence. Bacteria use monomeric adhesins/invasins or highly sophisticated macromolecular machines such as type III secretion systems and retractile type IV pili to establish a complex host/pathogen molecular crosstalk that leads to subversion of cellular functions and establishment of disease.

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The ClosTron: a universal gene knock-out system for the genus Clostridium.

John T. Heap, Oliver Pennington, Stephen Cartman … (2007)

Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.

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A mouse model of Clostridium difficile-associated disease.

Xinhua Chen, Kianoosh Katchar, Jeffrey Goldsmith … (2008)

Infection with Clostridium difficile causes nosocomial antibiotic-associated diarrhea and colitis. Hamsters historically have been used to investigate disease pathogenesis and treatment, but are not ideal models because of the lack of hamster-specific reagents and genetically modified animals, and because they develop fulminant disease. The aim of this study was to establish a mouse model of antibiotic-induced C. difficile-associated disease (CDAD) that more closely resembles human disease. C57BL/6 mice were exposed to a mixture of antibiotics (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 3 days. Two days later, they were given injections of clindamycin and then challenged 1 day later with different doses of C. difficile. Mice that were exposed to antibiotics and then challenged with C. difficile developed diarrhea and lost weight. Disease severity varied from fulminant to minimal in accordance with the challenge dose. Typical histologic features of CDAD were evident. Oral vancomycin prevented CDAD in all mice, but 68% died from colitis after treatment was discontinued. All animals that survived an initial episode of CDAD showed no evidence of diarrhea or colitis after subsequent rechallenge with C. difficile. Different strains of C. difficile tested in the model showed different levels of virulence in mice. We have developed a mouse model of CDAD that closely represents the human disease. In light of the recent substantial increases in CDAD incidence and severity, this model will be valuable in testing new treatments, examining disease pathogenesis, and elucidating mechanisms of protective immunity.