Conjugated Linoleic Acid Reduces Cholera Toxin Production In Vitro and In Vivo by Inhibiting Vibrio cholerae ToxT Activity

Abstract

The severe diarrheal disease cholera is endemic in over 50 countries. Current therapies for cholera patients involve oral and/or intravenous rehydration, often combined with the use of antibiotics to shorten the duration and intensity of the disease. However, as antibiotic resistance increases, treatment options will become limited. Linoleic acid has been shown to be a potent negative effector of V. cholerae virulence that acts on the major virulence transcription regulator protein, ToxT, to inhibit virulence gene expression. ToxT activates transcription of the two major virulence factors required for disease, cholera toxin (CT) and toxin-coregulated pilus (TCP). A conjugated form of linoleic acid (CLA) is currently sold over the counter as a dietary supplement and is generally recognized as safe by the U.S. Food and Drug Administration. This study examined whether CLA could be used as a new therapy to reduce CT production, which, in turn, would decrease disease duration and intensity in cholera patients. CLA could be used in place of traditional antibiotics and would be very unlikely to generate resistance, as it affects only virulence factor production and not bacterial growth or survival.

FIG 1

Cultures grown under virulence-inducing conditions in the presence of CLA exhibit reduced virulence gene expression. (A) tcpA::lacZ expression was significantly lowered upon the addition of 32, 64, and 128 μM CLA. (B) CT expression was significantly lowered upon the addition of 32, 64, and 128 μM CLA. Statistical significance was determined using Student's t test. *, P < 0.001.

FIG 2

Effects of different CLA isoforms on ToxT activity. V. cholerae was grown under virulence-inducing conditions with the indicated isoforms of CLA present at 32 μM, except for ME-CLA, which was present at 640 μM. β-Galactosidase assays were performed to examine P_tcpA activity. Statistical significance of results compared to the results for the DMSO-only control (white bar) was determined by Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n.s., not significant.

FIG 3

CLA inhibits ToxT binding to P_tcpA DNA. (A) Autoradiograph of the results of MBP-ToxT binding reactions with P_tcpA DNA. Lanes 1 to 7 include DMSO, and lanes 8 to 14 include 32 μM CLA in DMSO. The autoradiograph of EMSAs presented is representative of three or more independent experiments. (B) Binding curve using densitometry of the experiment whose results are shown in panel A. GraphPad software analysis of the K_d indicates that CLA induces a significant reduction in the binding affinity of ToxT for its cognate DNA. K_d values are shown in the inset, with significant difference between the best-fit values indicated by an asterisk: *, P < 0.0001.

FIG 4

CT ELISA of rabbit ileal loop fluid produced in the presence and absence of CLA. Ten-centimeter ileal loops were injected with approximately 10⁶ CFU V. cholerae C6706, followed by a separate injection containing either the indicated percentage of CLA in 10% Kollidon or control carrier 10% Kollidon (denoted by a zero in the CLA row) in a 1-ml volume. Analyzed data are presented as the mean results ± standard deviations. Statistical significance was determined by using the χ² and Student t tests. P values of less than 0.05 were considered statistically significant. **, P < 0.005; ***, P < 0.0005.

FIG 5

Ileal loop fluid volumes produced by V. cholerae C6706 in the presence or absence of CLA. Ten-centimeter ileal loops were injected with approximately 10⁶ CFU V. cholerae C6706, followed by a separate injection containing either the indicated percentage of CLA in 10% Kollidon or control carrier 10% Kollidon (noted by a zero in the CLA row) in a 1-ml volume. Values are presented as the ratio of fluid volume in milliliters to the length of the loop in centimeters. ***, P < 0.0005. FA, fluid accumulation.