Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae

The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epi demic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repe ating subunits of TcpA pilin that form long fibres, which laterally associa te into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and f unction. Alterations in the first domain, termed the structural domain, res ult in altered pilus stability or morphology. Alterations in the second dom ain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to m ediate bacterial interaction through direct pilus-pilus contact required fo r microcolony formation and productive intestinal colonization. The importa nce of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient muta nts simultaneously restore pilus-mediated bacterial interactions. The alter ations resulting from the suppressor mutations also provide insight into th e molecular interactions between pilin subunits within and between pilus fi bres.