Peptide inhibitors of Vibrio cholerae virulence in a Caenorhabditis elegans model of cholera

Abstract

The ever increasing challenge of pathogenic bacteria becoming resistant to multiple antibiotics has spawned the search for new antibacterial drugs and new targets for antibacterial drugs. Virulence factors could be such new drug targets. Here I present an in vivo model of the infectious disease cholera using the bacteria grazing nematode Caenorhabditis elegans. I show that the non-pathogenic (CTX¯) Vibrio cholerae O1 El Tor strain 2740-80 is ingested by C. elegans and establishes a lethal infection in the intestinal tract of the worm. I found that the virulence of V. cholerae, determined as its ability to kill C. elegans, was induced by growth of the bacteria under anaerobic conditions. I further wanted to test the effect of small cyclic peptides in preventing the virulence of V. cholerae and thereby rescuing C. elegans from infection. The peptides were synthesized by using the SICLOPPS (split intein-mediated circular ligation of peptides and proteins) technology. This allowed for the synthesis of 21 amino acids long cyclic peptides, with a central sequence of six randomized amino acids, intracellular in V. cholerae. A library of an estimated 14,000 clones was constructed, each clone expressing a different peptide. In order to test the individual clones a killing/rescue assay in liquid medium was set up in 96-well microtiter plates. This simple system was used to screen 350 clones/peptides. None of the clones promoted survival of the worms, but I contribute this to the low number of clones tested. Despite the lack of a positive outcome, my experiments indicate that this assay could be used to screen for peptides that target bacterial virulence.