Inhibition of virulence potential of Vibrio cholerae by natural compounds

Abstract

The rise in multi-drug resistant Vibrio cholerae strains is a big problem in treatment of patients suffering from severe cholera. Only a few studies have evaluated the potential of natural compounds against V. cholerae. Extracts from plants like 'neem', 'guazuma', 'daio', apple, hop, green tea and elephant garlic have been shown to inhibit bacterial growth or the secreted cholera toxin (CT). However, inhibiting bacterial growth like common antimicrobial agents may also impose selective pressure facilitating development of resistant strains. A natural compound that can inhibit virulence in V. cholerae is an alternative choice for remedy. Recently, some common spices were examined to check their inhibitory capacity against virulence expression of V. cholerae. Among them methanol extracts of red chili, sweet fennel and white pepper could substantially inhibit CT production. Fractionation of red chili methanol extracts indicated a hydrophobic nature of the inhibitory compound(s), and the n-hexane and 90 per cent methanol fractions could inhibit >90 per cent of CT production. Purification and further fractionation revealed that capsaicin is one of the major components among these red chili fractions. Indeed, capsaicin inhibited the production of CT in various V. cholerae strains regardless of serogroups and biotypes. The quantitative reverse transcription real-time PCR assay revealed that capsaicin dramatically reduced the expression of major virulence-related genes such as ctxA, tcpA and toxT but enhanced the expression of hns gene that transcribes a global prokaryotic gene regulator (H-NS). This indicates that the repression of CT production by capsaicin or red chili might be due to the repression of virulence genes transcription by H-NS. Regular intake of spices like red chili might be a good approach to fight against devastating cholera.

Fig. 1

Effect of spices extracts and its purified compounds on cholera toxin production of V. cholerae. V. cholerae O1 El Tor variant strain CRC41 was cultured in AKI medium in the presence of extracts of various solvents from red chili methanol extract. Identified compounds (capsaicin and fatty acid mixture) after fractionation (by thin layer chromatography) of red chili methanol extract were also tested. Comparative inhibition of CT production by methanol extracts of sweet fennel and white pepper are also shown. The amount of CT production is represented by mean ± SD.

Fig. 2

Effect of capsaicin on inhibition of cholera toxin production (%) in various V. cholerae strains. The numbers below X-axis indicate strains belonging to O1 El Tor possessing ctxB of El Tor type (n=5), O1 El Tor variant possessing ctxB of classical type (n=12), O139 strains possessing ctxB of El Tor (n=1) and classical type (n=1), respectively, non-O1/non-O139 (NAG) possessing ctxB of El Tor type (n=2), O1 classical (cla) possessing ctxB of classical type (n=2). Each experiment was done in triplicate and the variation in CT inhibition is represented by mean × SD. ‘CT^ET’ and ‗CT^cla’ represent El Tor and classical type of CT, respectively

Fig. 3

Effect of capsaicin on transcription of various regulatory and virulence genes in V. cholerae strain. Transcription of virulence-related genes of V. cholerae O1 El Tor variant CRC41 strain was investigated by qRT-PCR assay in the presence of capsaicin (100 μg/ml). The relative transcription level of each gene was compared using recA gene as an internal control. ‘C’ indicates control value of target gene transcription without red chili methanol extract and capsaicin (arbitrarily taken as 1). Statistical significance of the observed differences was calculated using a two-sample t test. (*, P<0.05; **, P<0.01).

Fig. 4

Possible mechanisms of virulence repression of V. cholerae by capsaicin. The arrow shows activation of transcription of toxR/toxS and tcpP/tcpH by environmental signals, which subsequently activates that of ctx and tcpA through activation of transcriptional activator toxT. H-NS is a basal repressor of toxT, ctx and tcpA under non-permissive condition. Capsaicin directly or indirectly repressed transcription of toxT, ctx and tcpA but enhanced that of hns gene (indicated by dot).