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

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.