Fig 1.
Synthetic compound 2-oxo-4-phenyl-2,5-dihydro-3-furancarbonitrile (MW01) has structural similarities with natural products.
MW01 (A) is a synthetically produced compound with structural parallels to natural products, such as ralfuranones (B) and flavipesin (C).
Fig 2.
Impact of MW01 on sucrose fermentation by V. cholerae.
Assays were carried out at 30°C in pH-MMSuc and microtiter plates. Absorbance measurements (A615) reflect the ability of bacterial cells to transport and ferment sucrose. The result of one representative experiment is shown. Data are shown for a PTS mutant (ΔEI) negative control, a wild-type V. cholerae (WT) positive control, and WT in the presence of the compound. Bacteria were exposed to MW01 at concentrations varying from 0.002- to 0.124-mg/mL. Conditions were tested in triplicate in each experiment, and three experimental replicates were performed on separate days. To account for the variability of initial absorbance measurements, experimental data were normalized to the initial A615 for each well.
Fig 3.
MW01 induces V. cholerae biofilm formation without effect on total growth in LB.
V. cholerae cultures were grown in borosilicate tubes in LB broth supplemented with 0.2 mg/mL of MW01 (A–C) or 0.1 mg/mL of MW01 (D–F). After 18 hours of incubation at 27°C, biofilm cells were harvested and quantified for total growth (A and D), planktonic growth (B and E), and biofilm growth (C and F). Data are shown for a PTS mutant (ΔEI) positive control growth in LB, wild-type V. cholerae (WT) in LB alone, and WT in LB supplemented with the compound. * and Ø denotes statistical significance (p < 0.05) from WT and ΔEI control values respectively.
Fig 4.
MW01 impedes growth of V. cholerae in early stages (within the first four hours) of bacterial culture.
V. cholerae cells were first exposed to MW01 in LB broth for four hours at 27°C in a setup that mimicked the initiation of biofilm cultures in borosilicate tubes. Then, after identical series of ten-fold dilutions, 10 μL of each broth culture was plated on LB agar and incubated overnight at 37°C. Wild-type V. cholerae (WT) not exposed to MW01 served as control. * denotes statistical significance (p < 0.05) from control values.
Fig 5.
Impact of MW01 on V. cholerae growth in MMGlu.
Assays were carried out at 30°C in microtiter plates. Optical density measurements at 600 nm (OD600) reflect the ability of V. cholerae to grow (both as planktonic and biofilm cells) in minimal medium supplemented with 0.5% wt/vol glucose (MMGlu). Data are shown for a wild-type V. cholerae (WT) alone or in the presence of MW01, as (A) bacteria grow over time in 96-well plate cultures and (B) at the 24-hour mark of the growth culture. Bacteria were exposed to MW01 at concentrations varying from 0.001- to 0.200-mg/mL. Conditions were tested in triplicate in each experiment, and three experimental replicates were performed on separate days.
Fig 6.
Impact of MW01 concentration on V. cholerae biofilm formation.
(A) Wild type (WT) V. cholerae cultures were used to inoculate borosilicate tubes filled with 1 μL of working MW01 solutions, at concentrations ranging from 0.00625–0.1 mg/mL, and 300 μl of MMPyr broth. The resulting biofilm cells were harvested after 20 hours of incubation at 27°C, and their optical densities were determined at 600 nm (OD600). A PTS mutant (ΔEI) served as positive control for biofilm growth. Stars indicate measurements that were statistically significantly different from WT biofilm formation in MMPyr alone. (B) WT V. cholerae biofilms forming at the air-liquid interface in borosilicate culture tubes while growth medium changes coloration in presence of MW01.
Fig 7.
MW01 reacts with pyruvate to form a distinctive blue colored product.
MW01 (0.1 mg/mL) in water and 0.5 wt/vol % aqueous solutions pyruvate and carbohydrates (mannose, fructose, and glucose).
Fig 8.
Proposed scheme showing the catalytic role of pyruvate in the reaction with MW01 (1).
Only in the presence of pyruvate does the methylene (δ 5.71, 2H, s) undergo abstraction resulting in proposed blue pigment 2.
Fig 9.
Schematic diagram of the PTS cascade.
MW01 interaction with pyruvate, upstream of the phosphor-transferase cascade, would likely impact the regulation of Vibrio biofilm formation and other bacterial regulatory networks through variation of the PEP:pyruvate ratio and modification of the phosphorylation state of the PTS enzymes. This hypothetical scenario however does not preclude the possibility of the pyruvate-MW01 complex forming in media solution and inducing biofilm formation through yet undetermined interactions with Vibrio cells.