Clostridium difficile, a spore-forming anaerobic Gram-positive bacillus, is the cause of a spectrum of gastrointestinal illness ranging from mild diarrhe though pseudomembranous colitis, and toxic megacolon. Alarmingly, the incidence, severity and mortality of C. difficile colitis have all increased significantly in the past twenty years. Th mechanism of C. difficile toxicity is well-characterized but no vaccine against C. difficile infecton exists and our knowledge about the interactions of C. difficile with its host is limited. One possible target for the host immune system are the Type IV pili of C. difficile. Type IV Pili (T4P) are hair-like surface appendages produced by many species of pathogenic Gram negative bacteria which play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, horizontal gene transfer and in numerous instances are essential for virulence. T4Ps are composed exclusively or primarily of many copies of pilin protein, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. This project aims to characterize the structure and functional significance of the proteins that make up the Type IV pili of C. difficile. T4Ps are produced by many species of pathogenic Gram-negative bacteria and are known to play a role in both cellular adhesion and colonization. T4Ps are composed exclusively or primarily of many copies of a pilin protein, tightly packed in a helix. Although better characterized in Gram-negative bacteria, T4P genes have been discovered in the genomes of all members of the Clostridium genus, including C. difficile. In collaboration with Dr. Michael Donnenberg, we have established structural investigations into six putative pilin genes identified in the C. difficile genome and have solved the structure of one PILA4, by x-ray crystallography. Rather than consisting of one ?-sheet surrounded by ?-helices, as is the case with all other known pilins, PILA4 contains a second ?-sheet at a ~70 angle to the helical pilin core. This second sheet and the surrounding helices give it a much more extended conformation from the core and this radically different exposed surface may play a role in cellular adhesion and possibly signaling of innate immune cells. We have used this structure in combination with data from other sources to model the structure of a pilus composed of PilA4. We have also grown isotopically-labeled samples of another pilin, PilA2, and have collected data using NMR spectroscopy to being assigning the resonances to determine the structure using distance and relaxation rates from NMR. Functional studies of PILA4 are currently underway while structural investigations into other pilin proteins continue both by x-ray crystallography and NMR.
Clostridium difficile is a bacterium which can become invasive, leading to an infection of the colon which is difficult to treat and it is likely that soe of that virulence is due to the ability of C. difficile to form appendages called Type IV pili (T4P). The goal of this project is to determine the structures of pilin proteins, the subunits that form T4P and to understand how they come together to form pili and what parts of human cells those pili interact with. There is currently no vaccine for C. difficile and antimicrobials are frequentl ineffective;making it particularly important to learn more about how C. difficile colonizes its human host.