Isolating and Purifying Clostridium difficile Spores

Edwards, Adrianne N.; McBride, Shonna M.

doi:10.1007/978-1-4939-6361-4_9

Cited by 65 publications

(60 citation statements)

References 34 publications

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“…C. difficile spores were obtained following standard procedures as previously described (Edwards and McBride, ). Briefly, C. difficile strains were grown overnight in TY broth with 10 μg ml –1 thiamphenicol.…”

Section: Methodsmentioning

confidence: 99%

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

Ransom

Kaus

Tran

et al. 2018

Molecular Microbiology

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Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σ , which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σ production by engineering cells to express tcdR from a tetracycline-inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σ and CodY, revealed neither is required for bimodal toxin gene expression. However, σ biased cells toward the Toxin-ON state, while CodY biased cells toward the Toxin-OFF state. Finally, toxin gene expression was observed in sporulating cells. We conclude that (i) toxin production is regulated by a bistable switch governed by σ , which only accumulates to high enough levels to trigger toxin gene expression in a subset of cells, and (ii) toxin production and sporulation are not mutually exclusive developmental programs.

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Section: Methodsmentioning

confidence: 99%

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

Ransom

Kaus

Tran

et al. 2018

Molecular Microbiology

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“…Hamsters treated with clindamycin, but not inoculated with C. difficile spores, served as negative controls. Spores were prepared as previously described Edwards and McBride, 2016;Woods et al, 2016). Following inoculation, hamsters were monitored for signs of disease, such as diarrhoea or wet tail, weight loss and lethargy.…”

Section: Hamster Studiesmentioning

confidence: 99%

Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis

Nawrocki

Wetzel

Jones

et al. 2018

Environmental Microbiology

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Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane-derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907-CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.

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“…difficile spores were purified as described elsewhere [46]. Spore suspensions were prepared by plating a 1:100 dilution of an overnight culture onto a 70:30 medium (63 g Bacto peptone (BD Difco), 3.5 g proteose peptone (BD Difco), 0.7 g ammonium sulfate (NH4)2SO4, 1.06 g Tris base, 11.1 g brain heart infusion extract (BD Difco) and 1.5 g yeast extract (BD Difco) for 1 L) and incubating it for 5 days at 37°C under anaerobic conditions [47]. After incubation, plates were removed from the chamber and the surface was scraped up with ice-cold sterile water.…”

Section: Methodsmentioning

confidence: 99%

Nasal immunization with the C-terminal domain of BclA3 induced specific IgG production and attenuated disease symptoms in mice infected withClostridioides difficilespores

Maia

Reyes-Ramírez

Pizarro-Guajardo

et al. 2020

Preprint

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Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance a strong immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets of new therapeutic treatments. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C.difficile spores. Here we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a well-established mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some CDI symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization route need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

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Isolating and Purifying Clostridium difficile Spores

Cited by 65 publications

References 34 publications

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis

Nasal immunization with the C-terminal domain of BclA3 induced specific IgG production and attenuated disease symptoms in mice infected withClostridioides difficilespores

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