Abstract
Vibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTXΦ, that transmits the cholera toxin-encoding genes. This toxigenic conversion of V. cholerae has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTXΦ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. To gain insight into the TolA-pIII complex, we developed a bacterial two-hybrid approach, named Oxi-BTH, suited for studying the interactions between disulfide bond-folded proteins in the bacterial cytoplasm of an Escherichia coli reporter strain. We found that two of the four disulfide bonds of pIII are required for its interaction with TolA. By combining Oxi-BTH assays, NMR, and genetic studies, we also demonstrate that two intermolecular salt bridges between TolA and pIII provide the driving forces of the complex interaction. Moreover, we show that TolA residue Arg-325 involved in one of the two salt bridges is critical for proper functioning of the Tol-Pal system. Our results imply that to prevent host evasion, CTXΦ uses an infection strategy that targets a highly conserved protein of Gram-negative bacteria essential for the fitness of V. cholerae in its natural environment.
Highlights
Vibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTX⌽, that transmits the cholera toxin-encoding genes
The TolAIII domains from V. cholerae and from E. coli were fused to the T18 domain in the pUT18 vector, whereas the pIII-N1 domains from M13 and CTX phages were fused to the T25 domain in the pKT25 vector
Constructs were introduced into the E. coli BTH101 strain, and co-transformants were tested on MacConkey plates
Summary
Vibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTX⌽, that transmits the cholera toxin-encoding genes. This toxigenic conversion of V. cholerae has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTX⌽ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. The bacterium produces CT and assembles new phage particles (carrying the ctxAB genes) that will be secreted in the environment, and it may convert nonpathogenic V. cholerae cells to pathogenicity. The N-terminal (N1) and the central domains (N2) are exposed at the capsid surface, the C-terminal domain (N3) anchors the pIII protein to the phage particle through hydrophobic interactions (4 – 6)
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