Exploring and exploiting the Vibrio cholerae Type IV pilus

The rise in antibiotic resistance coupled with a dearth of new antibiotics poses a significant threat to global health. Gram-negative bacterial pathogens are inherently resistant to large antibiotics such as vancomycin owing to their outer membrane barrier. The goal of my thesis project is to explore the potential of a bacterial transport machinery, the Type IV pilus (T4P), to act as an antibiotic delivery system. T4P are long, thin, retractile polymers of the major pilin protein expressed on the surfaces of many bacterial pathogens including Vibrio cholerae, enterotoxigenic Escherichia coli (ETEC) and Neisseria gonorrhoeae. Minor pilin proteins form a priming complex that initiates pilus formation and are situated at the tip of T4P where they function in adhesion and DNA uptake. The minor pilin TcpB forms a homotrimer at the tip of the V. cholerae T4P and is the receptor for the filamentous bacteriophage CTXΦ. Other T4P bind to extracellular DNA, most likely via their tip proteins, as a first step in natural transformation. I hypothesize that TcpB and other tip-associated pilus proteins can be targeted by carrier molecules comprised of antibody fragments (Fabs) or DNA aptamers to deliver antibiotics into the periplasm of bacterial pathogens via T4P retraction, thereby bypassing the outer membrane. To this end, I adopted a two-prong approach to find potential carriers: (1) I screened DNA aptamer libraries with recombinant TcpB through Systematic Evolution of Ligands by EXponential enrichment (SELEX); and (2) I characterized TcpB-specific phage displayed Fabs as binders of recombinant TcpB and the native TcpB at the tip of the pilus. This work resulted in several TcpB-specific Fabs capable of binding to the V. cholerae T4P tip and blocking CTXΦ transduction, which have potential as antibiotic carriers.