Title

Functional and biochemical characterization of pili in Corynebacterium diphtheriae

Date of Completion

January 2008

Keywords

Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

A variety of multi-subunit protein polymers on the bacterial cell surface known as pili or fimbriae play a pivotal role in the colonization of specific host tissues by many pathogens. Unlike gram-negative bacteria, the gram-positive pili are assembled by a distinct mechanism involving transpeptidases called sortase and this mechanism is conserved among gram-positive bacteria. Sortases cross-link individual pilin monomers and ultimately join the resulting covalent polymer to the cell wall peptidoglycan. A typical pilus consists of a major shaft protein and either one or two accessory minor pilins. Although much is known about the functional role of pili in pathogenesis in gram-negative bacteria, not much is known about the role of these pili in gram-positive bacteria, in particular in Corynebacterium diphtheriae which harbors three immunologically distinct pili. Functional and biochemical studies revealed the role of these pili in adhesion of the bacterium to host epithelial cells and demonstrated that these pili exhibit tissue tropism. Further analysis determined that the accessory pilins mediate this adhesion and are displayed on the cell surface even in the absence of the major shaft pilin. The current model of pilus assembly suggests that it is a biphasic process where polymerization catalyzed by the pilus specific sortase precedes the cell wall anchoring step which is catalyzed by the housekeeping sortase. Molecular genetic and biochemical studies demonstrated that SpaB, a minor pilin serves as a molecular switch between the polymerization and cell wall anchoring phase. Further analysis of the pilus assembly site revealed that the pilus specific sortase interacts with the housekeeping sortase to bring about pilus assembly. Using biochemical and functional studies, this thesis reveals the role of pili in corynebacterial attachment to host tissue and also dissects the pilus assembly site to gain insights into the assembly process. ^