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Infection and Immunity, April 2007, p. 1946-1953, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01435-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Effect of Fatty Acids and Cholesterol Present in Bile on Expression of Virulence Factors and Motility of Vibrio cholerae{triangledown}

Arpita Chatterjee,1 Pradeep K. Dutta,2 and Rukhsana Chowdhury1*

Biophysics Division,1 Medicinal Chemistry Division, Indian Institute of Chemical Biology, Calcutta 700 032, India2

Received 7 September 2006/ Returned for modification 9 November 2006/ Accepted 16 January 2007


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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.


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INTRODUCTION
 
Vibrio cholerae, a noninvasive enteric bacterium, is the causative agent of the diarrheal disease cholera. Cholera continues to cause devastating outbreaks, particularly in the developing world, resulting in more than 100,000 deaths every year, and the case fatality ratio may exceed 20% in affected populations (29). The pathogenicity of V. cholerae is largely due to the production of cholera toxin (CT) and a toxin-coregulated pilus (TCP), thought to be essential for colonization of the intestinal epithelium by the bacterium (13). Expression of CT, TCP, and several other virulence factors is coordinately controlled by the hierarchical expression of regulatory proteins comprising the ToxR regulon (14), in which the inner membrane DNA binding proteins ToxR and TcpP (7, 18) activate expression of ToxT, a transcriptional regulator that is required for the expression of ctxAB and tcpA as well as several other virulence genes (3). The ToxR regulon is strongly influenced by physicochemical parameters/factors such as temperature, osmolarity, pH, amino acids, and bile, which exert their effects at different levels of the regulatory cascade (16, 25).

Bile, a heterogeneous mixture of conjugated and unconjugated bile acids, bile pigments, inorganic salts, cholesterol, phospholipids, and probably other unidentified components, is secreted into the lumen of the duodenum from the gall bladder through the bile duct and is inevitably encountered by all enteric bacteria in their human hosts (9). The role of bile in normal gastrointestinal physiology is to aid the emulsification of lipids and also to protect the host from bacteria, due to the detergent-like activity of the amphipathic bile acids. However, enteric bacteria have evolved to survive in the presence of bile and many have further adapted to recognize bile as a signal indicative of their entry into the human host and can respond by inducing the production of virulence and other factors to facilitate survival in the host (5). Several studies have indicated that bile exerts pleiotropic effects on V. cholerae. Growth of V. cholerae in the presence of bile affects expression of virulence factors, induction of efflux pumps, motility, biofilm formation, and production of the outer membrane proteins OmpU and OmpT. Expression of the major virulence factors CT and TCP was repressed (6, 23), the AcrAB efflux pump was induced (2), motility was enhanced (6), biofilm formation was facilitated (12), and intracellular production of OmpU was stimulated with concomitant repression of OmpT synthesis (22) in V. cholerae grown in the presence of bile. Paradoxically, it has recently been reported that unlike crude bile, pure bile acids, unconjugated as well as glyco- and tauroconjugated, do not repress production of virulence factors and can actually stimulate CT production in a ToxR-dependent but ToxT-independent manner (11), suggesting that crude bile contains some component other than bile acids that represses virulence gene expression. It was in this context that the present study was designed to fractionate crude bile and identify the component(s) responsible for the repression of virulence gene expression and the enhancement of motility in V. cholerae. The results obtained indicated that unsaturated fatty acids present in bile inhibit the expression of virulence factors and that both unsaturated fatty acids and cholesterol enhance the motility of V. cholerae.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. The V. cholerae O1 strains O395, JJM43 (toxR) (27), and O395H29 (hns) (15) were used in the present study. The strains were grown under permissive conditions (LB, pH 6.6, 30°C) (18). In some experiments, bile fractions or fatty acids were added to the growth medium. Since the fractions and fatty acids were dissolved in methanol, calculated amounts of methanol were added to the control cultures in these experiments.

Fractionation of bile. Ox bile (15 g, catalog # 9875; Sigma) was dissolved in 50 ml of water and extracted successively with 200 ml each of petroleum ether (40 to 60°C), ethyl acetate, and butanol. Each extract was evaporated under reduced pressure. About 2 g petroleum ether extract, 3.5 g ethyl acetate extract, and 6.5 g butanol extract were obtained from 15 g bile. All solvents were redistilled prior to use.

TLC. The petroleum ether-, ethyl acetate-, and butanol-extracted fractions of bile were dissolved in methanol-chloroform, and the components were separated by thin-layer chromatography (TLC) on silica gel plates (Merck) by use of a solvent system consisting of ethyl acetate:ethyl methyl ketone:formic acid:water (5:3:1:1 [vol/vol]) for the ethyl acetate and butanol fractions, while a petroleum ether:diethylether:acetic acid (90:10:1 [vol/vol]) solvent system was used for the petroleum ether fraction. Spots were visualized by spraying with Lieberman-Burchard reagent, and plates were subsequently heated at 120°C for 5 min.

Silicic acid column chromatography. The petroleum ether fraction of bile (300 mg) was applied to a preequilibrated silicic acid (12-g) column (1 cm by 15 cm) and eluted successively with increasing concentrations of ethyl acetate in petroleum ether. Each fraction was examined by TLC as described above. Fractions with similar TLC profiles were pooled and dried under reduced pressure. Fractions eluted with petroleum ether:ethyl acetate (1:1 [vol/vol]) yielded a sticky mass (200 mg) that gave a single spot on TLC (spot I). Fractions eluted with petroleum ether:ethyl acetate (3:7 [vol/vol]) yielded a white solid that gave a distinct spot on TLC (spot II). It was crystallized from methanol, and the melting point was determined to be 146°C. The two fractions were analyzed further by 1H nuclear magnetic resonance (NMR) and gas chromatography (GC).

NMR. NMR spectra were recorded on a Bruker DPX-300 machine with tetramethyl silane as an internal standard.

GC and GC-MS. The fatty acid mixture obtained by silicic acid column chromatography was converted to methyl esters by use of dry methanol. Briefly, the fatty acid mixture (30 mg) was dissolved in dry methanol (5 ml), a 10% solution of acetyl chloride in methanol (5 ml) was added, and the mixture was kept at room temperature overnight. It was then partitioned between water and ether. The ether layer was washed first with 10% NaHCO3 solution (50 ml) and then with water, dried under nitrogen, and suspended in dry n-hexane for GC. Individual fatty acid methyl esters were separated by GC using an Agilent 6890 GC fitted with a flame ionization detector. The peaks were detected and estimated using a Hewlett-Packard 3398A GC Chemstation. For resolution, an HP-5 fused silica column (0.32 mm [inside diameter] by 30 m) was used. Nitrogen was used as a carrier gas with a flow rate of 1.4 ml/min, and the column oven temperature was programmed at 160°C for a 2-min hold followed by an increase at 3°C/min to a 220°C 18-min hold. The temperatures of the injection port and detector were 240°C and 300°C, respectively. GC-mass spectrometry (MS) was performed on a Shimadzu GC-MS-QP 5050A machine using a fused silica HP-5 MS capillary column (0.25 mm [inside diameter] by 30 m) with the same temperature program as used for GC and electron impact ionization at 70 eV with an ion source temperature of 200°C. Helium was used as the carrier gas.

Determination of MICs. The MICs of fatty acids were determined by serial twofold dilutions of the compounds in LB medium by use of an inoculum of 5 x 104 exponential-phase cells per milliliter. Turbidity (optical density) at 600 nm was measured after a 16-h incubation at 37°C with aeration. An optical density at 600 nm of less than 0.05 was considered as negative.

RNA isolation and real-time reverse transcription-PCR (RT-PCR). For isolation of RNA, V. cholerae strains were grown to the late logarithmic phase (7 x 108 to 109 CFU ml–1) in LB (pH 6.6) at 30°C. Total RNA was extracted and purified using guanidinium isothiocyanate (1). The RNA was treated with RNase-free DNase I (1U/µg, amplification grade; Invitrogen) in the presence of an RNase inhibitor (RNasin; GIBCO BRL). First-strand cDNAs were synthesized using a SuperScript first-strand synthesis system (Invitrogen) as per the manufacturer's instructions. Quantitative real-time PCR was performed in triplicate using a 7500 sequence detection system (Applied Biosystems). Amplifications were performed for the genes of interest and for 16S rRNA with SYBR green PCR master mix (Applied Biosystems). The V. cholerae genome sequence database (www.tigr.org) (8) was used to design the gene-specific primers for the genes of interest and 16S rRNA as previously described (24). PCR conditions consisted of AmpliTaq Gold activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s and annealing/extension at 60°C for 2 min. A dissociation curve was generated at the end of each cycle to verify that a single product was amplified using software provided with the 7500 sequence detection system. The change in SYBR green fluorescence was monitored by the system software, and the cycle threshold above background for each reaction was calculated. From the cycle threshold values, the levels of expression of the genes of interest and 16S rRNA relative to those for a control specified in the text were calculated. The statistical significance of the observed differences was calculated using a two-sample t test. A P value of <0.05 was considered significant.

GM1 ELISA. Since ganglioside GM1 is the cell surface receptor for CT, the binding of CT to GM1 immobilized on microtiter plates is widely used as an assay for CT (GM1 enzyme-linked immunosorbent assay [ELISA]). CT level was estimated for culture supernatants or sonicated cell pellets of V. cholerae grown in LB (pH 6.6) at 30°C in the absence or presence of 0.1% bile fractions, 0.03% fatty acids, or 0.04% cholesterol by GM1 ELISA. Control experiments in which spent culture supernatants of V. cholerae grown under permissive conditions, containing about 2 µg CT ml–1, were incubated without or with 0.1% bile fractions, 0.03% fatty acids, or 0.04% cholesterol were also performed, and no difference in the amounts of CT was detected for these samples by GM1 ELISA. Polyclonal rabbit serum directed against pure CT (Sigma) was used as the primary antibody. Anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Jackson Laboratories) was used as the secondary antibody. Dilutions of CT of known concentrations were used to estimate the amounts of CT in the samples.

Swarm plate assay. V. cholerae cells were stabbed into semisolid LB agar plates (pH 6.6) containing 0.3% agar (Difco). The plates were incubated at 30°C for 16 to 18 h, and swarm diameters were measured. Comparison among results obtained from assays performed in the absence or presence of bile fractions, fatty acids, or cholesterol was made by the two-sample t test.


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RESULTS
 
Fractionation of bile. An aqueous solution of ox bile was extracted successively with petroleum ether, ethyl acetate, and butanol, and the effect of each extract on CT production in V. cholerae strain O395 was examined (Fig. 1A). When grown under permissive conditions (LB medium, pH 6.6, at 30°C) (18), strain O395 produced about 2 µg CT in culture supernatants corresponding to 109 CFU. In contrast, when the cells were grown under identical conditions except for the presence of 0.1% petroleum ether fraction in the growth medium, practically no CT was detected in the culture supernatants (Fig. 1A) or sonicated cell pellets (data not shown). The presence of 0.1% ethyl acetate- or butanol-extracted fractions of bile in the growth medium had no significant effect on CT production (Fig. 1A). At a 0.1% concentration, none of the extracts had any effect on the growth of V. cholerae (Fig. 1B). TLC analysis indicated that the major components of the ethyl acetate and butanol fractions were conjugated and unconjugated bile salts (Fig. 2A), which have been reported to have no effect on the expression of virulence factors in V. cholerae (11). In view of the fact that the petroleum ether fraction of bile could completely inhibit CT production, this fraction was analyzed in greater detail.


Figure 1
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  		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.


Figure 2
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  		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.

Identification of components of the petroleum ether fraction of bile. TLC of the petroleum ether fraction indicated the presence of two distinct spots, spots I and II (Fig. 2B). Comparison with the TLC spots obtained using a pure cholesterol and fatty acid mixture (Fig. 2B), suggested that spot I might contain fatty acids while spot II might be due to cholesterol. To confirm the identity of the components of spot I and spot II, the petroleum ether fractions were separated by chromatography on silicic acid columns, and fractions corresponding to each spot were pooled as described in Materials and Methods and examined by NMR. The 1H NMR spectrum of the fraction corresponding to spot I showed peaks at {delta} 0.93 [t, -CH3], 1.25 [s, -(CH2)n-], 2.04 [m, -CH2-CHFormula CH-], 2.35 [m,-CH2-COO-], 2.77 [m, -CHFormula CH-CH2-CHFormula CH-], and 5.35[m, -CHFormula CH], indicating the presence of fatty acids. The 1H NMR spectrum of the fraction corresponding to spot II showed three proton singlets at {delta} 0.67 and 1.01 characteristic of C-18 and C-19 methyl groups, respectively, of a steroid moiety, besides a number of signals at 0.85 to 0.92 corresponding to other methyl groups. The NMR spectrum also showed signals at {delta} 3.5 [m, >CH-OH] and {delta} 5.35 [m, >CFormula CH-]. These signals indicated that the compound might be a sterol. Finally, the compound was identified as cholesterol by comparison of its 1H NMR and electron impact ionization-MS spectra (M+ 386) with those of a standard cholesterol sample. Thus, NMR data confirmed the results suggested by TLC analysis that a mixture of fatty acids was present in the fraction corresponding to spot I and that cholesterol was the only component identified in the fraction corresponding to spot II.

The fatty acid-containing fraction could completely inhibit the production of CT in V. cholerae, but the cholesterol fraction had no effect (Fig. 3A). The effect of fatty acid and cholesterol fractions on the expression of the ctxAB gene encoding CT was also examined by quantitative real-time RT-PCR. RNA was isolated from V. cholerae grown in the absence or in the presence of these fractions, and the amount of ctxAB-specific mRNA in each culture was estimated. Practically no ctxAB expression was detected for cells grown in the presence of the fraction containing fatty acids. However, cholesterol had no significant effect (P ≥ 0.05) on ctxAB expression (Fig. 3B).


Figure 3
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  		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.

Next, the individual fatty acids present in the fatty acid fraction were identified. The fraction was methylated, and the fatty acids present in the mixture and their relative abundances were determined from GC analysis (Fig. 4 and Table 1) and confirmed by GC-MS. The effect of each of these individual fatty acids on CT production and the expression of virulence genes of V. cholerae was next examined.


Figure 4
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  		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.


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  		TABLE 1. Fatty acids present in bile and their relative amounts obtained by GC analysis

Effect of fatty acids on expression of virulence genes of V. cholerae. The MICs of the major saturated and unsaturated fatty acids present in bile (Table 2) indicated that the growth of V. cholerae was considerably more sensitive to unsaturated fatty acids than to saturated fatty acids, as has been reported for other bacteria (31). The concentration at which none of the fatty acids examined had any detrimental effect on growth of V. cholerae was determined to be 0.03%; thus, all fatty acids were used at this concentration in subsequent experiments. The growth curve for V. cholerae in the presence of 0.03% oleic acid is shown in Fig. 1B. To examine the effect of the fatty acids on CT production, V. cholerae was grown in LB (pH 6.6) containing 0.03% palmitic, stearic, oleic, arachidonic, or linoleic acid, and CT levels were estimated for the cultures. Practically no CT was detected in cultures grown in the presence of oleic, linoleic, and arachidonic acids, while palmitic and stearic acids had no significant effect on CT production (Fig. 5A). Thus, saturated fatty acids had no effect on CT production, while unsaturated fatty acids almost completely inhibited CT production in V. cholerae.


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  		TABLE 2. Sensitivity of V. cholerae to fatty acidsa


Figure 5
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  		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.

Next, RNA was isolated from V. cholerae grown in the presence of a saturated fatty acid (palmitic acid) or an unsaturated fatty acid (linoleic acid), and the amounts of ctxAB-specific mRNA from these cultures were compared to those from cultures grown without fatty acids (Fig. 5B). Analysis of real-time RT-PCR results indicated no statistically significant difference (P = 0.1) in levels of ctxAB expression between cultures grown with palmitic acid and those grown without. However, ctxAB expression was reduced by more than 30-fold (P = 0.001) in cells grown in the presence of linoleic acid (Fig. 5B), although the amounts of 16S rRNA present in each RNA population were comparable (data not shown). Similar results were obtained with other unsaturated fatty acids present in bile, arachidonic acid and oleic acid (data not shown). The minimum concentration at which the unsaturated fatty acids could completely inhibit ctxAB expression was 0.015% (data not shown).

Since ctxAB expression is coordinately regulated with expression of the tcpA gene (27), the effects of palmitic acid and linoleic acid on tcpA gene expression were also examined. Real-time RT-PCR analysis indicated that palmitic acid had practically no effect on tcpA expression (P = 0.1), while linoleic acid significantly reduced tcpA expression (P ≤ 0.005) (Fig. 5B). Thus, unsaturated fatty acids repress expression of ctxAB and tcpA, the two major virulence genes of V. cholerae.

Expression of ctxAB and tcpA is coordinately regulated by the transcriptional activator ToxT (3). Since both ctxAB expression and tcpA expression were reduced in V. cholerae grown in the presence of unsaturated fatty acids, toxT expression was examined in these cells. Comparable levels of toxT expression (P = 0.1) were detected between cultures grown with and those grown without linoleic acids (Fig. 5B). Thus, in spite of normal expression of the transcriptional activator toxT, expression of the ToxT-activated genes ctxAB and tcpA was shut off in the presence of unsaturated fatty acids. It may be mentioned in this context that a similar pattern of expression of virulence genes and their regulator ToxT was observed for V. cholerae grown in the presence of bile (6, 23).

Role of H-NS in the fatty acid-mediated repression of virulence genes. It has been reported that the global prokaryotic gene regulator H-NS can bind to and repress expression from the ctxAB and tcpA promoters and that these genes are constitutively expressed under nonpermissive conditions of temperature, pH, and oxygen concentration in V. cholerae hns strains (15, 19). To examine whether the repression of ctxAB and tcpA by unsaturated fatty acids was mediated by H-NS, the effect of linoleic acid on ctxAB and tcpA expression was examined for the V. cholerae hns mutant strain O395H29 (15). Although linoleic acid strongly inhibited expression of ctxAB and tcpA in the wild-type V. cholerae strain O395, it had practically no effect on ctxAB and tcpA expression in the hns mutant strain O395H29 (P > 0.05) (Fig. 5B). When the strain O395H29 was complemented with plasmid pDIA562 carrying the full-length V. cholerae hns gene (28), ctxAB and tcpA gene expression was inhibited by linoleic acid to a level similar to that seen for wild-type strain O395 (Fig. 5B), suggesting that the phenotypic effects of the hns mutation on linoleic acid repression of ctxAB and tcpA expression were indeed due to disruption of hns and was not due to an unrecognized secondary mutation. Taken together, these results strongly suggested that H-NS represses expression of the two major virulence genes of V. cholerae in the presence of unsaturated fatty acids. To examine if ToxR/ToxT is required for expression of the virulence genes in the hns mutant strain grown in the presence of unsaturated fatty acids, a toxR hns double mutant of V. cholerae O395 was constructed by homologous integration of the suicide vector pGP704 (18), carrying an internal segment of the hns gene into the chromosomal hns gene of the toxR mutant strain JJM43 (27). The toxR hns mutant strain was designated V. cholerae O395HT. Although no expression of ctxAB and tcpA in the presence or absence of linoleic acid was observed for the V. cholerae toxR mutant strain JJM43, which contained a functional hns gene, significant expression of ctxAB and tcpA was detected for the toxR hns mutant strain O395HT even in the presence of linoleic acid (Fig. 5B).

Effect of fatty acids on motility of V. cholerae. In view of the fact that motility of V. cholerae was significantly increased in cells grown in the presence of bile (6), the effects of different components of bile on V. cholerae motility were examined by swarm plate assays. The results obtained indicated that the swarm diameter of strain O395 in the presence of a 0.1% petroleum ether fraction of bile (3.7 ± 0.5 cm) was about 140% of that in the absence of the fraction (2.7 ± 0.3 cm). The difference was statistically significant (P = 0.001). The ethyl acetate and butanol fractions of bile had no statistically significant effect on motility (P ≥ 0.05; data not shown). Since the petroleum ether fraction contained a mixture of fatty acids and cholesterol, the effects of these compounds on motility were examined (Fig. 6). When motility was assayed with swarm plates containing 0.03% linoleic acid, a statistically significant increase (P = 0.001) of 130% in the swarm diameter of V. cholerae O395 was consistently observed (Fig. 6). The average swarm diameter increased from 2.7 cm to 3.5 cm in the presence of linoleic acid. Similar results were obtained with oleic and arachidonic acids. For V. cholerae, motility is dependent on a single polar flagellum. The flagellar genes are organized in a regulatory cascade with the transcriptional activator flrA at the top of the hierarchy (21). Real-time RT-PCR analysis indicated a statistically significant 1.6-fold increase (P = 0.001) in flrA expression when V. cholerae was grown in the presence of linoleic acid (Fig. 7).


Figure 6
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  		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).


Figure 7
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  		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.

Role of H-NS and ToxR/ToxT in fatty acid-mediated enhancement of motility. Although H-NS represses the expression of many genes involved in bacterial response to environmental stresses, it positively regulates the expression of some flagellar genes whose products are essential for motility (20, 26). Indeed, the V. cholerae hns mutant strain was about twofold less motile than wild-type strain O395 (Fig. 6). To examine if H-NS has a role in the unsaturated fatty acid-dependent enhancement of V. cholerae motility, the effect of linoleic acid on the motility of the V. cholerae hns mutant strain O395H29 was examined. The swarm diameter of strain O395H29 was increased to about 130% (P ≤ 0.05) in the presence of 0.03% linoleic acid (Fig. 6). Thus, linoleic acid exerts comparable effects on the wild-type and hns mutant strains of V. cholerae, indicating that fatty acids enhance V. cholerae motility by an H-NS-independent mechanism. This result was supported by the observation that linoleic acid enhancement of motility was also observed for strain O395H29 complemented with plasmid pDIA526 carrying the V. cholerae hns gene (Fig. 6).

It has been demonstrated that the ToxR/ToxT system has a negative regulatory effect on motility (4), the mutant strain JJM43 lacking both ToxR and ToxT being about 133% (P = 0.001) as motile as the parent wild-type strain, O395 (Fig. 6). Although linoleic acid significantly increased the motility of wild-type strain O395, no statistically significant linoleic acid-dependent enhancement of motility was observed for the mutant strain JJM43 (Fig. 6). Taken together, these results suggest that in strain O395, unsaturated fatty acids might enhance motility by rendering ToxR/ToxT nonfunctional, thus overcoming the repressive action of these proteins on motility. However, in the toxR mutant strain JJM43, since ToxR/ToxT is absent, the motility of the strain is high and linoleic acid has no further effect on motility. Whether unsaturated fatty acids affect other regulators of motility is not yet known.

Cholesterol enhances motility of V. cholerae. In view of the fact that the petroleum ether fraction of bile greatly enhances V. cholerae motility, the effect of cholesterol, which together with fatty acids constitutes the petroleum ether fraction, on the motility of V. cholerae was also examined. At a concentration of 0.04%, cholesterol had no effect on growth or production of virulence factors in V. cholerae; however, the motility of the cholesterol-grown cells was increased to about 150% (P = 0.001; Fig. 6). A 2.5- to 3-fold increase in flrA expression was also observed for these cells (Fig. 7). The cholesterol-mediated increase in motility was observed for both the hns mutant strain O395H29 and the toxR mutant strain JJM43 (Fig. 6), indicating that H-NS and ToxR have no role in the process.


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DISCUSSION
 
In the course of infection, the enteric pathogen V. cholerae encounters changing physicochemical, nutritional, and other parameters in the different niches of the intestinal tract. It is now evident that these microenvironments impose a spatiotemporal control on the expression of virulence genes and other adaptive systems of V. cholerae at different stages of the pathogenesis cycle (17). Bile is recognized by V. cholerae as an environmental cue characteristic of the host intestinal lumen, to which the bacterium responds by the induction of pleiotropic effects, including repression of virulence gene expression and enhancement of motility. It has been hypothesized that increased motility might drive the bacteria into the mucus gel lining the intestine and that as the bacterium swims to a deeper location in the mucus, the bile concentration will decrease and the inhibition of ToxT activity will be relieved, resulting in expression of the TCP required for effective colonization (16, 23). In this study we present evidence that the effects of crude bile on V. cholerae are exerted by unsaturated fatty acids and cholesterol present in bile and examine the roles of different regulatory proteins in these processes. By extraction of ox bile with solvents of different polarities and analysis by TLC, NMR, GC, and GC-MS, fatty acids and cholesterol were identified as minor components of bile, while conjugated and unconjugated bile acids were doubtlessly the major components. The fatty acid fraction was comprised of palmitic and stearic acids (saturated fatty acids) and oleic, linoleic, and arachidonic acids (unsaturated fatty acids). The effects of each fatty acid and of cholesterol on the expression of virulence genes and motility were examined.

Cholesterol and the saturated fatty acids had no effect on the production of virulence factors in V. cholerae, but the unsaturated fatty acids present in bile, arachidonic, linoleic, and oleic acids, drastically repressed the expression of the major virulence genes, ctxAB and tcpA (Fig. 3B and 5B). No difference in the expression of toxT, encoding the direct transcriptional activator of ctxAB and tcpA, was detected between cells grown in the absence and those grown in the presence of unsaturated fatty acids (Fig. 5B). We next considered the possibility that the histone-like nucleoid-structuring protein H-NS, which has been implicated in the silencing of a large number of genes of diverse bacterial species, including V. cholerae (10, 19), might be involved in the fatty acid-dependent repression of ctxAB and tcpA. Indeed, the repression was completely abolished in a V. cholerae hns mutant (Fig. 5), suggesting that H-NS represses ctxAB and tcpA expression in the presence of unsaturated fatty acids. Is ToxR/ToxT required for the activation of ctxAB and tcpA in the hns mutant strain in the presence of unsaturated fatty acids? Since high levels of ctxAB and tcpA expression were observed for a toxR hns double mutant strain in the absence as well as the presence of unsaturated fatty acids, it would be reasonable to hypothesize that, as has been reported for other environmental conditions (19), in the absence of the negative regulator H-NS the positive regulators ToxR/ToxT are not necessary for ctxAB and tcpA gene expression in the presence of unsaturated fatty acids. It may be noted in this context that although it has earlier been demonstrated that H-NS can bind directly to ctxAB, tcpA, and toxT promoters and repress expression of these genes (15, 30), interestingly, results presented here suggest that H-NS selectively represses ctxAB and tcpA expression but has no effect on toxT expression in cells grown in the presence of unsaturated fatty acids.

Virulence gene expression and motility are known to be oppositely regulated in V. cholerae (4). Indeed, although unsaturated fatty acids repress expression of ctxAB and tcpA, the motility of the cells was increased in the presence of these compounds due to the induction of flrA, the first gene in a regulatory cascade that controls motility in V. cholerae (21). H-NS appears to be a positive regulator of motility in V. cholerae, since the motility of the V. cholerae hns mutant strain was lower that that of the wild-type parent strain (Fig. 6). On the other hand, it has been previously reported that activation of the ToxR regulon represses motility, although it is not clear whether the repression is mediated by ToxR or by ToxT (4). The role of these positive and negative regulators in the enhancement of motility by fatty acids has been examined in this study. If fatty acids facilitate the H-NS induction of motility, then in an hns mutant no fatty acid-mediated enhancement of motility would have been observed. However, motility of the hns mutant strain O395H29 was increased 150% in the presence of fatty acids, similar to what was seen for the wild-type strain (Fig. 6) suggesting that H-NS has no role in the fatty acid induction of motility. The role of ToxR/ToxT in the fatty acid enhancement of motility was next considered. Since these regulators exert negative modulatory effects on motility (4), it is attractive to hypothesize that the role of fatty acids is to render ToxR/ToxT nonfunctional, thus relieving the repression of motility. If that were indeed the case, fatty acids should have no effect on the motility of strain JJM43, in which both ToxR and ToxT are lacking. Indeed, the motility of strain JJM43 was not significantly affected by unsaturated fatty acids. However, since motility is known to be a multifactorial process controlled by many different regulators, the possibility also exists that unsaturated fatty acids may affect other regulators of motility.

Interestingly, cholesterol, another minor component of bile, also induces flrA expression and increases the motility of V. cholerae. Whereas flrA expression was increased less than twofold in the presence of fatty acids, a more-than-threefold increase was observed in the presence of cholesterol (Fig. 7). However, a cholesterol-dependent increase in motility was observed for both the hns and toxR mutant strains of V. cholerae, unlike the fatty acid-dependent motility enhancement, which was observed only for the hns mutant strain (Fig. 7). These results suggested that the enhancement of motility by cholesterol might not be dependent on the negative regulator ToxR/ToxT or on the positive regulator H-NS but might involve an as-yet-unknown regulatory system.

It is generally believed that susceptibilities to cholera vary widely among individuals and are markedly influenced by blood group antigens as well as predisposing factors such as low gastric acidity and malnutrition. In view of the multiple effects exerted by bile on V. cholerae, it is reasonable to assume that the composition and the amount of bile in the host intestine might also influence the outcome of V. cholerae infection. There are two fundamentally important functions of bile in all species. First, it is critical for digestion and absorption of fats and fat-soluble vitamins, and second, it provides a route for the elimination of many waste products from the body. Secretion into bile is a major route for eliminating excess cholesterol and fatty acids, which are virtually insoluble in aqueous solution but are soluble in the presence of the amphipathic bile acids in bile. The amounts of fatty acids and cholesterol present in bile might vary widely among individuals, and even in the same individual, variations may occur at different times, depending upon diet and other factors. It is likely that these variations might affect the incidence and severity of cholera following infection of an individual by V. cholerae.


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ACKNOWLEDGMENTS
 
We thank all members of the Biophysics and Medicinal Chemistry Divisions for cooperation, encouragement, and helpful discussions during the study and I. Guha Thakurta, P. Majumdar, and Subodh Roy for excellent technical support. We are grateful to J. J. Mekalanos, Harvard Medical School, Boston, MA, for the generous gift of strain JJM43 and to A. K. Sen, Medicinal Chemistry Division, for assistance with GC and GC-MS.

The work was supported by a research grant from the Network Program (SMM 003), Council of Scientific and Industrial Research (CSIR), Government of India. A.C. is grateful to CSIR for a research fellowship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Biophysics Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700 032, India. Phone: 91 33 473 0350. Fax: 91 33 473 5197. E-mail: rukhsana{at}iicb.res.in. Back

{triangledown} Published ahead of print on 29 January 2007. Back

Editor: V. J. DiRita


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Infection and Immunity, April 2007, p. 1946-1953, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01435-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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