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.

Glucose Metabolism by Escherichia coli Inhibits Vibrio cholerae Intestinal Colonization of Zebrafish

“…This ion uptake is essential to replace the lost electrolytes and water caused by the voluminous diarrhea caused by cholera toxin (23). On the other hand, there are concerns that the glucose content in ORS might worsen the disease by increasing the expression of toxic genes in El Tor strains (22). Here we reversed the inductive effect of glucose on El Tor strains by using acid-producing E. coli.…”

“…Additionally, acetoin production offers V. cholerae cells a survival advantage during infection by downregulating the host innate immune responses (21). Another report showed that inhibiting acetoin production could be an antibacterial mechanism for V. cholerae elimination (22). Previous work explored another way to reduce the growth of El Tor strains in vitro by using a coculture of E. coli that is acid producing in the presence of glucose in the medium (13).…”

“…This is the first observation that glucose feeding to zebrafish can significantly reduce the colonization of classical strain O395 and increase the colonization of El Tor strain N16961. Other groups previously showed the effect of glucose on V. cholerae colonization in the infant mouse model, but the glucose treatment of V. cholerae was in vitro before the infection (22). In a clinical scenario, pathogenic bacteria cannot be treated before infection; glucose must be an effector of colonization during the treatment.…”

Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae

“…Bacterial SR has been a target to be inhibited. Chemical compounds that can suppress (p)ppGpp production, such as relacin (74) and iMAC (75), can potentially be used as antibiotic adjuvants. We anticipate that experimental data provided in the current study will stimulate future investigations that eventually help us come up with better strategies to combat bacterial infections, including one by the deadly enteric pathogen V. cholerae.…”

Alterations in glucose metabolism in Vibrio cholerae serogroup O1 El Tor biotype strains

“…This change in V. cholerae population has remained a contradiction, of which a difference in glucose metabolism between the 2 biotype strains has been widely accepted to be a cause 5 . El Tor biotype strains use glucose by neutral fermentation, whereas classical biotype strains perform acidic fermentation of glucose, which is considered an advantage for the former with regard to survival in a host and perhaps in nature 5,6 .…”