Type IV pili (T4P) are filamentous structures formed by polymerization of thousands of pilin subunits. They are involved in diverse bacterial functions including twitching motility, DNA uptake, phage attachment, and adherence to biotic and abiotic surfaces. In some bacterial models, T4P are virulence factors involved in host cell adherence. Besides the major structural pilin subunit, T4P possess minor pilin-like proteins. T4P are expressed in many Gram-negative bacteria and were recently found in Gram-positive bacteria. Enteropathogenic Escherichia coli (EPEC) and Clostridium difficile (C. difficile) are two enterovirulent bacteria that cause diarrhoeagenic illness by different mechanisms. EPEC express T4P called Bundle forming pili (BFP) that are involved in the initial adherence of the bacteria to host-cells. C. difficile has the genes required for T4P but their expression or function are unknown. In my study I used a combination of genetic and imaging techniques to investigate T4P expressed by both EPEC and C. difficile. In EPEC, I investigated the role of three pilin-like proteins BfpI, BfpJ, and BfpK in the BFP structure and function. I found that all three proteins are co-assembled in BFP. I also found that BfpI is involved in EPEC adhesion to HEp-2 cells since the phenotype of localized adherence was lost in EPEC expressing BFP without BfpI. I also found that BfpJ is involved in regulating pilus extension since BFP lacking BfpJ forms only short pilus structures. Finally, although BfpK is co-assembled with BfpA within the pilus filament, I could not detect any phenotypic differences between BfpK-deficient and wild-type BFP.
In the study involving C. difficile I found growing conditions, which allowed the bacteria to express T4P bundles in the hypervirulent R20291 strain. I then used pilin and pilin-like mutants to discover that only PilA1 and PilU proteins are essential for pilus expression in C. difficile and that PilA1 and PilJ proteins are present within the pilus filament. Furthermore, searching for a role of T4P in colonization, I found that C. difficile T4P is not involved, at least in mice, since there was no difference between wild-type and T4P mutant strains in infecting mice.