Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Joseph W. St. Geme III
The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children. Although usually present as a commensal organism, it can sometimes breach the epithelial barrier, enter the bloodstream, and spread hematogenously to sites of infection. Adherence to epithelial cells is thought to be the first step in K. kingae colonization of the oropharynx and a prerequisite for invasive disease. Understanding the molecular mechanisms of K. kingae adherence will generate a broader understanding of the pathogenesis of K. kingae disease and may identify novel targets for therapeutic disruption of the involved processes. Previous work has established that type IV pili and the pilus-associated proteins PilC1 and PilC2 are required for K. kingae adherence; however, the molecular mechanisms by which PilC1 and PilC2 promote adherence have remained elusive. We hypothesized that K. kingae PilC1 and PilC2 directly bind to epithelial cells and sought to define their roles in adherence and other type IV pilus phenotypes. In this work, we evaluated surface piliation, adherence, twitching motility, and natural competence in a panel of PilC mutant strains. PilC1 and PilC2 were required for adhesive fibers, while purified PilC1 and PilC2 exhibited saturable binding to epithelial cell monolayers. The proteins showed distinct global protein structures and adhesive phenotypes. Strains producing PilC1 or PilC2 truncates containing only the C-terminal domain produced surface fibers that lacked adhesive activity, while purified PilC1 and PilC2 N-terminal domain fragments exhibited saturable binding to epithelial cells. Furthermore, we identified a predicted metal ion-dependent adhesion site (MIDAS) motif in the N-terminal domain of K. kingae PilC1 and hypothesized that it promoted type IV pilus adhesive activity. We mutated critical residues of the MIDAS motif and then evaluated K. kingae type IV pilus phenotypes. Our results showed that the MIDAS mutant had defects in adherence, twitching motility, and natural competence, and purified PilC1 MIDAS mutant protein was unable to bind to epithelial cells. Taken together, our data show that PilC1 and PilC2 are adhesins that differentially promote bacterial adherence by directly interacting with epithelial cells using their N-terminal domains, with PilC1 adopting a eukaryotic MIDAS motif for adhesive interactions.
Sacharok, Alexandra Louise, "Kingella Kingae Pilc1 And Pilc2 Are Adhesins With Distinct Roles In Type Iv Pilus-Mediated Processes" (2021). Publicly Accessible Penn Dissertations. 4959.