Function and Vaccine Potential of a Clostridium difficile Type IV Pilus Subunit

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DESCRIPTION (provided by applicant): Clostridium difficile is a Gram-positive, spore-forming, obligate anaerobe that has become the leading cause of nosocomial diarrhea in the United States. Infection with the bacterium has a variety of manifestations, ranging from asymptomatic colonization of the colon to copious diarrhea, pseudomembranous colitis, and death. Antibiotic treatment for infection is available, although treatment can fail and patients can relapse. Other available therapies include fecal transplant and, in very serious cases, colectomy. C. difficile infection results in 14,000 deaths per year; thus, a better understanding of its pathogenesis as well as improved preventative measures are urgently necessary. Although the exact sequence of events in initial colonization with C. difficile is still under investigation, evidence from othr intestinal pathogens suggests that attachment to epithelial cells is a requisite step in pathogenesis. Genes for components of a Type IV pilus (T4P), a type of bacterial appendage used in adhesion and motility, were recently identified in the C. difficile genome. The main body of the T4P is composed of pilins; the major structural subunit is the major pilin, whereas other, similarly structured proteins termed minor pilins or pilin-like proteins, are present at lower abundances and have various roles in pilus biogenesis. The C. difficile genome encodes nine specific pilins or pilin-like proteins. This proposal focuses on the function and vaccine potential of one such protein, PilJ. I hypothesize that PilJ is a minor pilin, and is present at a lower abundance than a predicted major pilin, PilA1; I will use quantitative immunoblotting and qRT-PCR to test this hypothesis. Furthermore, I hypothesize that PilJ is the T4P adhesin, and that it interacts with a specific receptor on the host cell. To identify this receptor, I will take two different approaches; one involves cross-linking the pilin to its receptor, while the other makes use of a human haploid cell line. Finally, I hypothesize that immunization with PilJ and adjuvant will be protective against challenge with C. difficile in a murine model of C. difficile infection. Immunization of mice and rabbits with PilJ produces a strong and specific response to the protein; I predict that this response will be protective. Outcomes for this experiment will be mortality, morbidity, histopathology, and terminal antibody titer. These results will illuminate th role of PilJ in the C. difficile T4P and in interactions between the T4P and the host. Additionally they offer an exciting new target for a C. difficile vaccine.