Electrostatic interactions between the CTX phage minor coat protein and the bacterial host receptor TolA drive the pathogenic conversion of Vibrio cholerae

Abstract

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 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 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 in its natural environment.

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

“…Enzymes (NewEngland Biolabs) were used according to the manufacturer's instructions. Plasmids were constructed using standard cloning technics, as previously described (21,41). Mutations on pBAD-tolQR and pOKtolA plasmids were performed by Quick-change site-directed mutagenesis using complementary pairs of oligonucleotides (listed in Table S2) and Pfu Turbo polymerase.…”

“…Indeed, TolAIII is sufficient for binding Fd phage protein pIII-N1 in vitro (19,23) and in a bacterial 2-hybrid assay (21). It was also shown that overexpressing the isolated TolAIII domain in the host periplasm disturbs the normal functioning of the Tol system, and decreases cell susceptibility to phage infection, possibly by sequestering the phage in the periplasm and preventing the infection to proceed to the next step (7).…”

“…Once in the periplasmic space, filamentous phages require both TolA, TolQ and TolR for efficient infection (translocation step) (7,8,12,17,18). A direct interaction between TolA Cterminal domain (TolAIII) and the phage pIII-N-terminal domain (pIII-N1) has been documented (19)(20)(21)(22)(23), while the role of TolQ and TolR proteins remains unclear. TolA, TolQ and TolR proteins are part of the Tol-Pal system, a pmf-dependent molecular motor conserved in Gram-negative bacteria.…”

Bacterial outer membrane constriction

“…TolB is essential for viability in P. aeruginosa (Lo Sciuto et al ., ), also TolA and TolQ are putatively essential in the closely related P. putida (Dennis et al ., ). The TM helix of TolA appears to be essential for viability of V. cholerae as the full length gene cannot be deleted while TolA II–III can be (Houot et al ., ). Evidence suggests that Tol–Pal plays a similar role in each of these species, as mutation gives rise to the pleiotropic tol – pal OM phenotypes and cell division defects (Heilpern and Waldor, ; Llamas et al ., ; Lo Sciuto et al ., ; Houot et al ., ).…”

Similarities and Differences between Colicin and Filamentous Phage Uptake by Bacterial Cells

“…The Tol system has been reported to be essential in many bacterial species, whereas it is dispensable in the E. coli K12 strain (62)(63)(64)(65)(66). In E. coli, deletion of any of the tol genes causes a pleiotropic phenotype.…”