The cyclic dipeptide cyclo(Phe-Pro) inhibits cholera toxin and toxin-coregulated pilus production in O1 El Tor Vibrio cholerae

Abstract

Cyclo(Phe-Pro) is a cyclic dipeptide produced by multiple Vibrio species. In this work, we present evidence that cyclo(Phe-Pro) inhibits the production of the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) in O1 El Tor Vibrio cholerae strain N16961 during growth under virulence gene-inducing conditions. The cyclo(Phe-Pro) inhibition of CT and TCP production correlated with reduced transcription of the virulence regulator tcpPH and was alleviated by overexpression of tcpPH.

FIG. 1.

cFP inhibits CT and TCP production. V. cholerae was cultured under AKI conditions in the presence of the indicated concentrations of cFP before CT and TcpA production was assayed. (A) Cholera toxin production. The results are the averages of the results of three or more independent experiments ± standard deviations. Statistical analysis was performed using a Tukey-Kramer multiple comparison test. *, P < 0.05; **, P < 0.01. (B) Results of TcpA Western blot analysis of whole-cell lysates.

FIG. 2.

cFP inhibits tcpPH, toxT, tcpA, and ctxAB transcription. V. cholerae containing the indicated promoter-lux reporters was grown under AKI conditions in the presence or absence of 1 mM cFP for 5 h, at which time luminescence production was quantified. (A) Effects of 1 mM cFP on gene expression. RLU, relative light units. (B) Results of TcpP Western blot analysis of strain N16961 grown in the presence or absence of 1 mM cFP. (C) Effects of 1 mM cFP on aphA and aphB expression at 5 h. MU, Miller units. The reported results are the averages of the results of three or more experiments ± standard deviations.

FIG. 3.

Overexpression of tcpPH and toxT alleviates cFP-dependent inhibition of CT and TCP production. V. cholerae containing the indicated expression vector was grown under AKI conditions in the presence or absence of 1 mM cFP. Arabinose (0.004%) was added to the AKI broth to induce expression of toxT and tcpPH. (A) Cholera toxin production. The CT results are the averages of the results of three experiments ± standard deviations. (B) Results of TcpA Western blot analysis of whole-cell lysates.