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Tackling a difficult bug

Researchers characterize spore formation in Clostridium difficile

Oeiras, 04.10.2013

Clostridium difficile is a nasty bacterium. Unable to survive in the presence of oxygen, it relies on the production of highly resilient spores to spread and cause infection. Till now no one knew how the sporulation process occurred, also because this bug is tough to work with in the lab, but researchers from the Microbial Development Lab and the Institute Pasteur, in Paris, developed new techniques to fully characterize the sporulation process in this important human pathogen. The work is published in two back-to-back papers in PLoS Genetics.

Clostridium difficile has been a largely overlooked bacterium but is the cause of an intestinal disease whose symptoms range from mild diarrhea to severe, potentially lethal inflammatory lesions such as pseudomembraneous colitis, toxic megacolon or bowel perforation. Recent years have seen the emergence of hypervirulent strains linked to increased incidence of severe disease, higher relapse rates and mortality making C. difficile the main hospital-acquired pathogen associated with antibiotic therapy. Infection often starts with the ingestion of spores, which germinate in the colon to establish a population of vegetative cells that will both produce two potent cytotoxins and more spores. The toxins are responsible for most of the disease symptoms, whereas the spores, which can remain latent in the gut, are both a transmission factor and a major cause of disease recurrence. Understanding how spores are formed in these bacteria will help develop strategies for diagnostic and therapy.

The inability of Clostridium to live in the presence of oxygen makes the whole manipulation in the lab extremely difficult. Researchers at the Microbial Development Lab developed a new set of strategies to follow the sporulation process. These include working with a new reporter protein that, unlike the commonly used Green Fluorecent Protein (GFP), fluoresces in the absence of oxygen. With this reporter, researchers were able to follow the expression of different sporulation genes in single cells throughout the sprorulation process. By combining this approach with traditional genetics, i.e. creating mutants for the main regulatory proteins in sporulation, it was possible to fully characterize the sporulation process, something that had never been done in Clostridium difficile.

The sporulation process in C. difficile resembles that of Bacillus subtilus, a related bacteria used as the model for studies of sporulation, suggesting that the top level of the sporulation network is conserved in evolution, using the same key regulatory proteins, and was established some 2.5 billion years ago. The process, however, is less tightly regulated in C. difficile. Researchers propose that this may be a general feature of spore formation in the more ancient Clostridia group of bacteria. The two studies also establish a platform for further studies of the developmental network in C. difficile, and in particular for inspecting the expression and function of the sporulation genes, many of which will be species-specific, and related to host colonization and transmission.


Original articles

PLoS Genet 9(10): e1003782. doi:10.1371/journal.pgen.1003782

The spore differentiation pathway in the enteric pathogen Clostridium difficile

Fátima C. Pereira1, Laure Saujet, Ana R. Tomé, Mónica Serrano, Marc Monot, Evelyne Couture-Tosi, Isabelle Martin-Verstraete, Bruno Dupuy, and Adriano O. Henriques


PLoS Genet 9(10): e1003756. doi:10.1371/journal.pgen.1003756

Genome-wide Analysis of Cell type-specific Gene Expression during Spore Formation in Clostridium difficile

Laure Saujet, Fátima C. Pereira, Monica Serrano, Olga Soutourina, Marc Monot, Pavel .V Shelyakin, Mikhail S. Gelfand, Bruno Dupuy, Adriano O. Henriques and Isabelle Martin-Verstraete

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