Cloning in an AviTag to Minor Pilins for Optimal Display during Antibody Library Screening

Abstract

Bacteria possess intricate systems of rapidly assembling and disassembling filaments called Type IV Pili (T4P), which are responsible for diverse functions including bacterial aggregation, twitching motility, and phage and DNA uptake. Each pilus is a polymer of thousands of copies of the major pilin and small numbers of minor pilins that have roles in assembly, retraction and pilus functions. In simple T4P systems minor pilins form a homotrimer that initiates pilus assembly and localizes to  the pilus tip. In the Vibrio cholerae toxin coregulated pilus, this minor pilin homotrimer binds to the CTXf phage, which is taken into the bacterium by pilus retraction. In complex T4P systems like those of Neisseria gonorrhoeae and Pseudomonas aeruginosa five different minor pilins form a homopentamer that primes pilus assembly and is involved in DNA binding and uptake. The adhesive and retractile features of T4P make them ideal targets for antibody carriers that can deliver antibiotics into the bacterial periplasm. The long term goal of this research is to exploit the T4P machinery as an antibiotic delivery system by selecting from diverse phage display library antibodies that bind to minor pilins at the tip of the pilus that can be used as antibiotic carriers. To this end, we cloned an Avitag onto the N-terminus of recombinant minor pilins CofB of enterotoxigenic Escherichia coli (ETEC) and TcpB of V. cholerae. The AviTag can be biotinylated using the biotin ligase BirA, allowing the minor pilins to be immobilized on streptavidin-coated plates in an orientation that mimics the pilus tip for antibody library screening.