Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System.

M Shamim Hasan Zahid,Shinji Yamasaki,Shah M Faruque,Atsushi Hinenoya,Shruti Chatterjee,Sharda Prasad Awasthi,Masahiro Asakura,Mahfuzar Rahman

doi:10.1371/journal.pone.0137529

M Shamim Hasan Zahid, Shinji Yamasaki + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0137529

Copy DOI

Abstract

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

Highlights

The current status of emergence of multidrug resistant (MDR) pathogenic bacteria has caused loss of the effectiveness of antimicrobial agents against them [1]
We tested the effect of anethole on the production of cholera toxin (CT) and toxin coregulated pilus (TCP) by culturing strains in presence of sub-bactericidal concentration of anethole
To select an optimal concentration of anethole that would not affect the bacterial growth, we tested the effect of anethole on the growth of 14 different V. cholerae strains and found that up to 100 μg/ml of anethole did not have any detectable effect on the growth of these strains (S1 Table)

Summary

Introduction

The current status of emergence of multidrug resistant (MDR) pathogenic bacteria has caused loss of the effectiveness of antimicrobial agents against them [1]. Multidrug resistance in toxigenic Vibrio cholerae, the causative agent of cholera epidemics is a growing concern. Over 200 different ‘O’ serogroups of V. cholerae have already been documented, only O1 (El Tor and classical biotypes) and O139 are responsible for cholera outbreaks [4]. The O1 El Tor biotype of V. cholerae is responsible for the ongoing 7th cholera pandemic, and this biotype replaced the classical biotype strain which caused the 6th cholera pandemic. Emerged V. cholerae O1 El Tor variant strains (possess some attributes of classical biotype including ctxB gene allele) produce more CT and cause more severe symptoms of diarrhea than prototype El Tor [6, 7]

Methods

Results

Conclusion