Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence

Abstract

To be successful pathogens, bacteria must often restrict the expression of virulence genes to host environments. This requires a physical or chemical marker of the host environment as well as a cognate bacterial system for sensing the presence of a host to appropriately time the activation of virulence. However, there have been remarkably few such signal-sensor pairs identified, and the molecular mechanisms for host-sensing are virtually unknown. By directly applying a reporter strain of Vibrio cholerae, the causative agent of cholera, to a thin layer chromatography (TLC) plate containing mouse intestinal extracts, we found two host signals that activate virulence gene transcription. One of these was revealed to be the bile salt taurocholate. We then show that a set of bile salts cause dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulfide bonds between cysteine (C)-207 residues in its periplasmic domain. Various genetic and biochemical analyses led us to propose a model in which the other cysteine in the periplasmic domain, C218, forms an inhibitory intramolecular disulfide bond with C207 that must be isomerized to form the active C207-C207 intermolecular bond. We then found bile salt-dependent effects of these cysteine mutations on survival in vivo, correlating to our in vitro model. Our results are a demonstration of a mechanism for direct activation of the V. cholerae virulence cascade by a host signal molecule. They further provide a paradigm for recognition of the host environment in pathogenic bacteria through periplasmic cysteine oxidation.

Fig. 1.

Bile salts activate virulence genes. (A) The V. cholerae virulence regulatory cascade. See text for details. (B and C) Mouse intestinal extracts were chromatographed on cellulose TLC plates and detected by UV (B) or overlaid with LB agar containing V. cholerae P_tcpA-lux reporter cells and then incubated anaerobically at 37 °C for 4 h. After incubation, the TLC plate was photographed in the dark using a LAS4010 ImageQuant and analyzed using Living Image 3.2 (C). (Inset) The chemical structure of VAI-1(TC). (D) A total of 100 µM synthetic bile salts (Sigma) was incubated with V. cholerae (P_tcpA-lux) anaerobically at 37 °C for 4 h. The luminescence of cells was read using a Bio-Tek Synergy HT spectrophotometer and normalized for growth against optical density at 600 nm and compared with equal amount by mass of purified VAF-1. (E) The corresponding cell-free culture fluids from (D) were assayed in GM1 ganglioside ELISAs for CT (32). (F) TcpA Western blot of El Tor O1 (C6706), El Tor O139 (MO10), and classical O395 grown in LB (El Tor) or LB pH 8.5 [classical, noninducing condition (34)] in the absence or in the presence of 100 µM TC for 5 h. Data are mean and SD of three independent experiments. CA, cholic acid; CDC, chenodeoxycholate; DC, deoxycholate; GC, glycocholate.

Fig. 2.

Bile salts regulate TcpP activity through its periplasmic domain. (A) The effect of bile salts on transcription of virulence regulators. V. cholerae containing promoter-lux transcriptional fusion plasmids were grown in LB in the absence or in the presence of 100 µM TC anaerobically at 37 °C until OD₆₀₀ = 0.2. Luminescence was measured and reported as fold induction (+TC/−TC). (B) E. coli containing P_ctx-lux reporter and either vector control or P_BAD-toxRS plasmids were grown in LB containing 0.01% arabinose in the absence or in the presence of 100 µM until OD₆₀₀ ≈ 0.2. Luminescence was then measured and reported as light units/OD₆₀₀. (C) E. coli containing P_toxT-lux reporter and either P_BAD vector control or indicated P_BAD-protein chimeras were grown in LB containing 0.01% arabinose in the absence or in the presence of 100 µM TC until OD₆₀₀ = 0.2. Luminescence was then measured and reported as light units/OD₆₀₀. C, cytoplasmic domain; P, periplasmic domain; _P, TcpP; _R, ToxR; T, transmembrane domain. Data are mean and SD of three independent experiments.

Fig. 3.

Bile salts mediate TcpP dimerization through cysteine-207. (A) Bile salts promote TcpP–TcpP interaction. Full-length TcpP and truncated/chimeric TcpP were fused with the T25 and T18 domains of adenylate cyclase (CyaA) from Bordetella pertussis, respectively, and the T25, T18 fusion pairs were introduced into E. coli cyaA mutants (17) or cyaA/dsbA double mutants (35). Cultures were grown at 30 °C for 8 h and β-galactosidase activity was measured and reported as Miller Units (36). The annotation is the same as in Fig. 2C. (B) V. cholerae ΔtcpPH (pBAD-tcpPH) containing P_toxT-lux reporter and WT or cysteine mutant tcpP under the control of the P_BAD promoter on plasmids were grown in LB containing 0.01% arabinose in the absence or in the presence of 100 µM TC until OD₆₀₀ ≈ 0.2. Luminescence was then measured and reported as light units/OD₆₀₀. Data are mean and SD of three independent experiments. (C) V. cholerae tcpP deletion mutants containing P_BAD-controlled plasmids harboring TcpP and its cysteine mutant derivatives fused with C-terminal FLAG tags were grown in LB containing 0.01% arabinose in the absence or in the presence of 100 µM TC. Then, 1-mg cell lysates were applied to a nonreducing SDS/PAGE (without DTT, Upper) or to a reducing SDS/PAGE (with 10 mM DTT in the loading buffer, Lower), and subjected to the Western blot using anti-FLAG antibody. D, dimer; M, monomer.

Fig. 4.

Sensing bile salts in vivo. (A) A working model: an intermolecular disulfide bond at C207 is critical for TcpP activity. In the absence of bile salts, an intramolecular disulfide bond forms between C207 and C218. Upon encountering bile salts, isomerization takes place to cause an intermolecular disulfide bond between two subunits of TcpP at C207. (B) In vivo competition assay using an infant mouse model. Six-day-old CD-1 infant mice were inoculated with the mixture of TcpP cysteine mutant and WT at 1:1 ratio with or without cholestyramine. After a 6-h period of colonization, intestinal homogenates were collected, and the ratio of mutant-to-WT bacteria was determined and normalized against input ratios. ***P < 0.001 (Student t test).