Selective and Efficient Elimination of Vibrio cholerae with a Chemical Modulator that Targets Glucose Metabolism.

Young Taek Oh,Sang Sun Yoon,Wontae Hwang,Hwa Young Kim,Eun Jin Kim,Hyoung Rae Kim,Dong Wook Kim,Junhyeok Go

doi:10.3389/fcimb.2016.00156

Abstract

Vibrio cholerae, a Gram-negative bacterium, is the causative agent of pandemic cholera. Previous studies have shown that the survival of the seventh pandemic El Tor biotype V. cholerae strain N16961 requires production of acetoin in a glucose-rich environment. The production of acetoin, a neutral fermentation end-product, allows V. cholerae to metabolize glucose without a pH drop, which is mediated by the production of organic acid. This finding suggests that inhibition of acetoin fermentation can result in V. cholerae elimination by causing a pH imbalance under glucose-rich conditions. Here, we developed a simple high-throughput screening method and identified an inducer of medium acidification (iMAC). Of 8364 compounds screened, we identified one chemical, 5-(4-chloro-2-nitrobenzoyl)-6-hydroxy-1,3-dimethylpyrimidine-2,4(1H,3H)-dione, that successfully killed glucose-metabolizing N16961 by inducing acidic stress. When N16961 was grown with abundant glucose in the presence of iMAC, acetoin production was completely suppressed and concomitant accumulation of lactate and acetate was observed. Using a beta-galactosidase activity assay with a single-copy palsD::lacZ reporter fusion, we show that that iMAC likely inhibits acetoin production at the transcriptional level. Thin-layer chromatography revealed that iMAC causes a significantly reduced accumulation of intracellular (p)ppGpp, a bacterial stringent response alarmone known to positively regulate acetoin production. In vivo bacterial colonization and fluid accumulation were also markedly decreased after iMAC treatment. Finally, we demonstrate iMAC-induced bacterial killing for 22 different V. cholerae strains belonging to diverse serotypes. Together, our results suggest that iMAC, acting as a metabolic modulator, has strong potential as a novel antibacterial agent for treatment against cholera.

Highlights

Acetoin, known as 3-hydroxy-2-butanone or acetyl methyl carbinol, is produced by various microorganisms in which acetoin often has physiological functions required for survival (Voloch et al, 1983; Yoon and Mekalanos, 2006; Vivijs et al, 2014a,b)
In the seventh pandemic N16961 El Tor strain, (p)ppGpp-mediated regulation is more important for maintaining bacterial viability than the quorum sensing (QS)-dependent regulation, which is controlled in an AphA-dependent manner (Oh et al, 2015)
N16961 and its alsS mutant were grown in LB or LB plus 1% glucose (LBG) with each chemical compound

Summary

Introduction

Known as 3-hydroxy-2-butanone or acetyl methyl carbinol, is produced by various microorganisms in which acetoin often has physiological functions required for survival (Voloch et al, 1983; Yoon and Mekalanos, 2006; Vivijs et al, 2014a,b). Because acetoin is a neutral fermentation end product and this biosynthetic reaction consumes intracellular protons, bacterial growth can occur on a glucose carbon source without pH decrease (Thomas et al, 2014). These findings indicate that acetoin production during fermentation plays an important physiological role in the adaptation to acidic stress, and it suggests a potential mechanism to control pathogenic bacteria like V. cholerae, that are known to be acid-sensitive (Merrell and Camilli, 2002)

Methods

Results

Conclusion