Vibrio campbelliichitoporin: thermostability study and implications for the development of therapeutic agents againstVibrioinfections

Abstract

AbstractVibrio campbellii(formerlyVibrio harveyi) is a bacterial pathogen that causes vibriosis, which devastates fisheries and aquaculture worldwide.V. campbelliiexpresses chitinolytic enzymes and chitin binding/transport proteins, which serve as excellent targets for antimicrobial agent development. We previously characterizedVhChiP, a chitooligosaccharide-specific porin from the outer membrane ofV. campbelliiBAA-1116. This study employed far-UV circular dichroism and tryptophan fluorescence spectroscopy, together with single channel electrophysiology to demonstrate that the strong binding of chitoligosaccharides enhanced thermal stability ofVhChiP. The alanine substitution of Trp136at the center of the affinity sites caused a marked decrease in the binding affinity and decreased the thermal stability ofVhChiP. Tryptophan fluorescence titrations over a range of temperatures showed greater free-energy changes on ligand binding (ΔG°binding) with increasing chain length of the chitooligosaccharides. Our findings suggest the possibility of designing stable channel-blockers, using sugar-based analogs that serve as antimicrobial agents, active againstVibrioinfection.