CTXφ Infection of Vibrio cholerae Requires the tolQRA Gene Products

Abstract

CTX is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTX. To test whether CTX also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTX uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTX entry. We could detect CTX infection of TCP ؊ V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTX infection. Thus, CTX, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.

Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces

“…TolA forms an inner membrane complex with TolQ, TolR, and TolB, encoded by the tolQRAB gene cluster conserved in Gram-negative bacteria (55) and is involved in outer membrane integrity, energy transduction, and cell division (56 -58). E. coli and V. cholerae tolQRA mutants are viable but are sensitive to detergents and antibiotics, suggesting a role for these proteins in maintaining outer membrane integrity (11,59,60). E. coli tolQRAB mutants are resistant to filamentous phage infection as well as to colicins, bacteriocidal molecules that are produced by some E. coli strains and act upon other E. coli strains (61,62).…”

“…TolA Is Not Required for CTX Binding to V. cholerae but Is Required for Uptake-To investigate the role of V. cholerae TolA in CTX infection, phage were incubated with V. cholerae strain O395 and with an O395-derived ⌬tolA deletion mutant, DH3 (11), and visualized by immunogold labeling and TEM. V. cholerae produce relatively low levels of CTX that are difficult to detect by TEM.…”

“…V. cholerae produce relatively low levels of CTX that are difficult to detect by TEM. Thus, we used the engineered fd phage fd⌬1-pIII CTX (15-274), which expresses CTX pIII domains N1 and N2 in place of the native fd N1N2, causing a switch in specificity from E. coli to V. cholerae (11). Plasmidexpressed fd⌬1-pIII CTX are produced in high levels from E. coli DH5␣-pir (53).…”

“…The core region of the CTX prophage contains genes cep, orfU, ace, zot, ctxA, and ctxB. Although not homologous in sequence, the cep, orfU, ace, and zot genes are similar in size and synteny to the M13 genes VIII, III, VI, and I, which encode the structural proteins pVIII, pIII, pVI, and pI, respectively (3,11). In M13, the major coat protein pVIII forms the long, cylindrical phage coat that packages the phage genome.…”

“…The CTX phage utilize a similar mechanism to infect V. cholerae, as both TCP and TolA are required for maximal infectivity (3,11). Heilpern and Waldor (25) generated recombinant fd phage in which CTX pIII domains N1, N2, or N1N2 were fused to the N terminus of fd pIII or to deletion mutants lacking N1N2 and assessed the ability of these hybrid phage to infect V. cholerae.…”