Effect of fatty acids and cholesterol present in bile on expression of virulence factors and motility of Vibrio cholerae

Abstract

Bile induces pleiotropic responses that affect production of virulence factors, motility, and other phenotypes in the enteric pathogen Vibrio cholerae. Since bile is a heterogeneous mixture, crude bile was fractionated, and the components that mediate virulence gene repression and enhancement of motility were identified by nuclear magnetic resonance, gas chromatography (GC), and GC-mass spectrometry analyses. The unsaturated fatty acids detected in bile, arachidonic, linoleic, and oleic acids, drastically repressed expression of the ctxAB and tcpA genes, which encode cholera toxin and the major subunit of the toxin-coregulated pilus, respectively. The unsaturated fatty acid-dependent repression was due to silencing of ctxAB and tcpA expression by the histone-like nucleoid-structuring protein H-NS, even in the presence of the transcriptional activator ToxT. Unsaturated fatty acids also enhanced motility of V. cholerae due to increased expression of flrA, the first gene of a regulatory cascade that controls motility. H-NS had no role in the fatty acid-mediated enhancement of motility. It is likely that the ToxR/ToxT system that negatively regulates motility is rendered nonfunctional in the presence of unsaturated fatty acids, leading to an increase in motility. Motility and flrA expression were also increased in the presence of cholesterol, another component of bile, in an H-NS- and ToxR/ToxT-independent manner.

FIG. 1.

Effect of bile fractions on growth and CT production. (A) V. cholerae O395 was grown in LB (pH 6.6) at 30°C in the absence (C) or in the presence of 0.1% petroleum ether fraction (PE), ethyl acetate fraction (EA), or butanol fraction (B) of bile. CT was estimated in culture supernatants and expressed as the amount obtained in culture supernatants corresponding to 10⁹ CFU. The data represent the averages of five independent experiments, and the error bars indicate standard errors of the mean. (B) Growth curves of V. cholerae O395 grown in LB (pH 6.6) at 30°C in the presence of 0.1% petroleum ether fraction, ethyl acetate fraction, or butanol fraction (Bu) of bile or in the presence of 0.03% oleic acids (OA). In a control experiment, V. cholerae was grown in LB (pH 6.6) at 30°C in the presence of calculated amounts of methanol.

FIG. 2.

TLC of bile fractions. (A) Lanes: CDC, standard sodium cholate and deoxycholate; EA, ethyl acetate fraction of bile; TC, standard sodium taurocholate; Bu, butanol fraction of bile; GC, standard sodium glycocholate; TDC, standard sodium taurodeoxycholate. (B) TLC of petroleum ether fraction of bile (lane PE) showing spots comigrating with standard fatty acid mixture (spot I) and standard cholesterol (spot II). Lane Ch, standard cholesterol; lane FA, standard oleic acid and palmitic acid mixture.

FIG. 3.

Effect of fatty acid and cholesterol fractions on CT production and ctxAB expression. The petroleum ether fraction of bile was further separated by silicic acid column chromatography into fractions containing fatty acids and cholesterol. V. cholerae O395 was grown in LB (pH 6.6) at 30°C (C) or in the presence of 0.05% fatty acid fraction (FA) or 0.04% cholesterol fraction (Ch). (A) CT was estimated in culture supernatants and expressed as the amount obtained in culture supernatants corresponding to 10⁹ CFU. The error bars indicate standard errors of the mean. (B) RNA was isolated from the cultures and real-time RT-PCR performed for quantitation of ctxAB expression. 16S rRNA expression was used as an internal control. The value for ctxA expression in strain O395 grown in LB without fatty acids was arbitrarily taken as 1. Results of three independent experiments are represented as means ± standard deviations (SD). Statistical significance of the observed differences was calculated using a two-sample t test. A P value of <0.05 was considered significant.

FIG. 4.

GC analysis of the fatty acids present in bile. GC spectra of methyl ester derivatives of the fatty acid mixture obtained by silicic acid column chromatography of the petroleum ether fraction of bile (bottom panel) and standard fatty acid methyl esters (top panel). Abbreviations: La, lauric acid; M, myristic acid; P, palmitic acid; L, linoleic acid; O, oleic acid; S, stearic acid; A, arachidonic acid.

FIG. 5.

Effect of fatty acids on virulence gene expression. (A) V. cholerae was grown in LB (pH 6.6) at 30°C in the absence (bar labeled C) or in the presence of 0.03% arachidonic acid (AA), linoleic acid (LA), oleic acid (OA), palmitic acid (PA), or stearic acid (SA). CT was estimated in culture supernatants and expressed as the amounts obtained in culture supernatants corresponding to 10⁹ CFU. The error bars indicate standard errors of the mean. (B) Real-time RT-PCR was performed for estimation of ctxAB, tcpA, and toxT expression with RNA isolated from V. cholerae O395, O395H29 (hns), O395H29 carrying plasmid pDIA562, and O395HT grown in LB (pH 6.6, 30°C) (dotted bars) or in LB containing either 0.03% linoleic acid (diagonally hatched bars) or 0.03% palmitic acid (grey bars). 16S rRNA expression was used as an internal control. The value for ctxA expression in strain O395 grown in LB without fatty acids was arbitrarily taken as 1. Results of three independent experiments are represented as means ± SD. A P value of <0.05 was considered significant.

FIG. 6.

Swarming of V. cholerae strains O395, JJM43 (toxR), O395H29 (hns), and O395H29 carrying pDIA562 on motility agar plates without (C) or with 0.03% linoleic acid (LA) or 0.04% cholesterol (Ch).

FIG. 7.

Effect of fatty acid and cholesterol on flrA gene expression. RNA was isolated from V. cholerae strain O395 grown in LB (pH 6.6) at 30°C (C) or in LB containing 0.03% of linoleic acid (LA) or 0.04% cholesterol (Ch) for the estimation of flrA expression. 16S rRNA expression was used as an internal control. The value for flrA expression in strain O395 (C) was arbitrarily taken as 1, and the data are presented as means ± SD.