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.

Keywords: Vibrio cholerae; acetoin; glucose metabolism; inducer of medium acidification (iMAC); mixed acid fermentation.

Figure 1

Schematic diagram of the experimental procedures to identify chemical compounds that inhibit acetoin fermentation. (A) The strategy of V. cholerae elimination using a chemical inhibitor that is capable of suppressing acetoin fermentation. (B) A high- throughput screening assay used to identify iMAC. Bacterial cells were inoculated in LB or LBG (LB containing 1% glucose) on a 96-well plate containing glass beads for effective aeration and were grown in aerobic conditions for 16 h. To verify the pH of culture supernatants, phenol-red was used as a pH indicator.

Figure 2

N16961, the seventh pandemic El Tor strain, showed compromised viability in glucose-rich conditions in the presence of iMAC. (A) The structure and IUPAC name of iMAC. (B) Bacterial cells were inoculated in LB or LBG with varying concentrations of iMAC and were grown for 16 h. Relative growth is calculated from OD₆₀₀ measurements. ^*p < 0.03 vs. OD₆₀₀ values of LBG-grown cultures (iMAC untreated control). Data is presented as the mean ± SD (error bars) determined from three independent replicate experiments. (C) Changes in cell viability after treatment with iMAC. Aliquots of each culture were sampled at 16 h post-inoculation and were 10-fold serially diluted to assess the number of colony-forming units (CFUs). (D) Scanning electron microscope (SEM) images of V. cholerae strains grown in LB or LBG, in both the presence and absence of 50 μM iMAC treatment. Cells were grown for 16 h prior to processing for SEM. The images were acquired at 25,000x magnification.

Figure 3

iMAC modulates glucose metabolism and results in high levels of organic acids and low levels of acetoin, a neutral fermentation product. (A) Metabolite and glucose levels in the culture supernatant were measured using HPLC as described in the Materials and Methods. ^*p < 0.0001, ^**p < 0.05 vs. metabolite levels that were measured in culture media of wild-type N16961 grown with LBG (iMAC untreated control). Non-detected values are displayed as “ND.” Data are presented as the mean ± SD (error bars) based on three independent replicate experiments. (B) Wild type N16961 strain was inoculated in phosphate-buffered (pH 7.5) or non-buffered LB/ LBG, respectively, either 50 μM iMAC treated or untreated, and were grown in aerobic conditions for 16 h. OD₆₀₀ values were expressed as relative growth. ^*p < 0.05 vs. OD₆₀₀ value grown in buffered LBG with iMAC. Three independent experiments were performed, and values of mean ± SD (error bars) are displayed in each bar. (C) The acetoin level in the culture supernatant was measured by Voges-Proskauer (VP) tests for bacterial culture supernatants. Bacteria were cultured in each media for 16 h. ^*p < 0.003 vs. acetoin levels in LBG grown cultures. (D) The promoter activity of the alsD (VC1589) gene in wild-type N16961 strain grown in LB, LBG, or LBG + 50 μM iMAC for 4 h. The reporter strain containing chromosomal lacZ reporter fusion was assayed in triplicate for β-galactosidase activity. Data are presented as the mean ± SD. ^*p < 0.002 vs. β-galactosidase activity of cells grown in LBG.

Figure 4

Effects of iMAC on (p)ppGpp-dependent acetoin production. (A) Intracellular (p)ppGpp levels were detected using TLC analysis. Wild-type N16961 and (p)ppGpp⁰ mutant (ΔrelA ΔspoT ΔrelV triple genes deletion mutant) were grown in LB or LB + 50 μM iMAC with P₃₂-orthophosphate for 4 h. Cellular extracts were prepared as described in the Materials and Methods and analyzed using TLC. (B) CT levels in the culture supernatants were measured by CT-ELISA. Wild-type N16961 and (p)ppGpp⁰ mutant were grown under two different CT-inducible culture conditions as described in Materials and Methods. ^*p < 0.003 vs. CT levels produced under grown in plain LB. ^**p < 0.03 vs. CT levels of N16961 produced under grown in LBT. Data presented as the mean ± SD based on three independent replicate experiments.

Figure 5

Effects of iMAC on V. cholerae in vivo infectivity. Infant mice (n > 8) were infected with 50 μL of each V. cholerae N16961 strain (1 × 10⁶ cells) suspended in indicated media supplemented with or without 50 μM iMAC. After 24 h, mice were sacrificed, and the entire intestines were extracted. (A) The number of viable cells was measured after 16 h by counting the number of viable bacterial cells recovered from the mouse intestine. The solid horizontal lines represent the geometric mean of each sample. ^*p < 0.001 vs. CFU from infection with LB, LB + iMAC, or LBG. Each symbol shows the value obtained from each mouse. (B) The fluid accumulation ratio was calculated as described in Materials and Methods. Data are presented as the mean ± SD based on three independent replicate experiments. ^*p < 0.001 vs. fluid accumulation ratio from uninfected control, ^**p < 0.004 vs. fluid accumulation ratio from infection with LB, LB+iMAC, or LBG.

Figure 6

A potential mechanism by which iMAC, acting as a ppGpp-dependent metabolic switch, induces inviability of V. cholerae. In the presence of glucose, V. cholerae normally metabolize glucose by the mixed acid fermentation during early stages. As growth proceeds, the environmental pH decreases as a result of the accumulation of organic acid metabolites. The glucose metabolic pathway then switches to acetoin fermentation to avoid lethal acidification. Previous work demonstrates that intracellular (p)ppGpp has a critical role in this glucose metabolic switch. Here, iMAC is characterized in terms of its potential role in inhibiting the accumulation of intracellular (p)ppGpp. Overall, iMAC successfully prevents the ppGpp-dependent glucose metabolic switch by inhibiting the accumulation of intracellular ppGpp. As a result, cells lose their viability as their environment becomes acidified as a result of uncontrolled organic acid fermentation in glucose-enriched conditions.