Bicarbonate Induces Vibrio cholerae virulence gene expression by enhancing ToxT activity

Abstract

Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO(2) into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.

FIG. 1.

Effect of bicarbonate on CT and tcpA::lacZ expression. Open bars, wild-type V. cholerae grown without bicarbonate; dark gray bars, wild-type V. cholerae grown with 0.3% bicarbonate; light gray bars, V. cholerae ΔtoxT mutant grown with 0.3% bicarbonate. (A) CT production by El Tor strain E7946. (B) β-Galactosidase produced from plasmid-borne tcpA::lacZ in El Tor strain E7946. (C) CT production by classical strain O395. (D) β-Galactosidase produced from chromosomal tcpA-lacZ in classical strain O395. Statistical significance was determined by Student's t test (*, P < 0.025; **, P < 0.005; ***, P < 0.0005). OD600, optical density at 600 nm; WT, wild type.

FIG. 2.

Bicarbonate does not increase toxT transcription. (A) β-Galactosidase produced from plasmid-borne toxT::lacZ in El Tor strain E7946. Light gray bars, V. cholerae ΔtoxR mutant grown without bicarbonate; black bars, V. cholerae ΔtoxR mutant grown with 0.3% bicarbonate; open bars, wild-type V. cholerae grown without bicarbonate; dark gray bars, wild-type V. cholerae grown with 0.3% bicarbonate. Statistical significance was determined by Student's t test (*, P < 0.02). WT, wild type. (B) RT-PCR to detect toxT mRNA in whole-cell RNA preparations. − RT, no RT was performed before PCR; + RT, RT was performed before PCR. Lane M contained molecular weight standards, and 4 hr and 6 hr indicate the time of growth in a stationary tube. AKI indicates addition of a shaking phase of growth. The presence or absence of bicarbonate in the growth medium is indicated below the gels.

FIG. 3.

Detection of ToxT protein in V. cholerae grown in the presence or absence of bicarbonate. ToxT protein was detected by Western blotting using polyclonal anti-ToxT antibodies. 6His-ToxT, purified His-tagged ToxT protein loaded as a control. (A) El Tor strain E7946. The presence or absence of bicarbonate in the growth medium is indicated above the lanes. E7946 ΔtoxT + pBAD-toxT indicates that the ΔtoxT strain was complemented in trans with pBAD-toxT and arabinose was included in the growth medium. (B) Classical strain O395. 4 hr and 6 hr indicate the time of growth in a stationary tube, and AKI indicates that a shaking phase of growth was added. Lane M contained protein molecular weight markers.

FIG. 4.

Effect of ToxT overproduction from pBAD-toxT on CT production in V. cholerae El Tor strain E7946. Open bars, bacteria grown without bicarbonate; gray bars, bacteria grown with 0.3% bicarbonate. Both strains were grown statically for 6 h in the presence of arabinose before a CT ELISA was performed. E7946 ΔtoxT + pBAD-toxT indicates that the ΔtoxT E7946 strain was complemented in trans with pBAD-toxT, and E7946 + pBAD33 indicates wild-type V. cholerae carrying the empty pBAD33 vector. Statistical significance was determined by Student's t test (*, P < 0.005). OD600, optical density at 600 nm.

FIG. 5.

Effects of the CA inhibitor EZA on tcpA::lacZ expression. Gray bars, dimethyl sulfoxide (DMSO) alone added to the growth medium; open bars, EZA dissolved in dimethyl sulfoxide added to the growth medium. (A) β-Galactosidase produced from plasmid-borne tcpA::lacZ in El Tor strain E7946. (B) β-Galactosidase produced from chromosomal tcpA-lacZ in classical strain O395. Statistical significance was determined by Student's t test (*, P < 0.015; **, P < 0.005).

FIG. 6.

Model for induction of virulence gene expression by bicarbonate in vivo. On the left, motile V. cholerae containing inactive ToxT protein enters the upper small intestine. In the center, V. cholerae in the intestinal lumen encounters bicarbonate, ToxT becomes active, and TCP production begins. On the right, bacteria entering the mucus layer encounter higher levels of bicarbonate, virulence genes are fully induced, and CT production begins. The gradient of increasing bicarbonate levels from the lumen to the mucosal surface is indicated by the triangle on the right.