Aggregation of Vibrio cholerae by Cationic Polymers Enhances Quorum Sensing but Overrides Biofilm Dissipation in Response to Autoinduction

Abstract

Vibrio cholerae is a Gram-negative bacterium found in aquatic environments and a human pathogen of global significance. Its transition between host-associated and environmental lifestyles involves the tight regulation of niche-specific phenotypes such as motility, biofilm formation, and virulence. V. cholerae's transition from the host to environmental dispersal usually involves suppression of virulence and dispersion of biofilm communities. In contrast to this naturally occurring transition, bacterial aggregation by cationic polymers triggers a unique response, which is to suppress virulence gene expression while also triggering biofilm formation by V. cholerae, an artificial combination of traits that is potentially very useful to bind and neutralize the pathogen from contaminated water. Here, we set out to uncover the mechanistic basis of this polymer-triggered bacterial behavior. We found that bacteria-polymer aggregates undergo rapid autoinduction and achieve quorum sensing at bacterial densities far below those required for autoinduction in the absence of polymers. We demonstrate this induction of quorum sensing is due both to a rapid formation of autoinducer gradients and local enhancement of autoinducer concentrations within bacterial clusters as well as the stimulation of CAI-1 and AI-2 production by aggregated bacteria. We further found that polymers cause an induction of the biofilm-specific regulator VpsR and the biofilm structural protein RbmA, bypassing the usual suppression of biofilm during autoinduction. Overall, this study highlights that synthetic materials can be used to cross-wire natural bacterial responses to achieve a combination of phenotypes with potentially useful applications.

Figure 1

Polymers enhance quorum sensing in Vibrio cholerae. Chemical structures of P1 (A) and P2 (B). N-SIM super-resolution image of LIVE/DEAD-stained V. cholerae A1552 clustered by P1 (C). V. cholerae El Tor strains A1552 (D, E, H) or E7646 (F, G, I) containing the luminescence reporter pBB1 were adjusted to an OD₆₀₀ of 0.2 following 16 h of growth and incubated in AMW alone (black) or AMW-containing polymer P1 (D, F) or P2 (E, G) at concentrations of 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL^–1. Luminescence was recorded every 30 min for 10 h and plotted as means ± SEM from at least three biological replicates. For the effect of polymers on bacterial viability to be tested, samples were removed after 10 h, serially diluted, and plated on LB (H, I).

Figure 2

Bacterial density shapes the kinetics of quorum induction in response to polymer. Cultures of V. cholerae A1552 containing pBB1 were grown for 16 h and diluted into AMW alone (black) or AMW containing 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL^–1 of P1 (A–C) or P2 (D–F). Bacterial densities were adjusted to result in OD₆₀₀ values of 0.5 (A, D), 0.05 (B, E), and 0.005 (C, F), respectively. Luminescence and OD₆₀₀ were recorded every 30 min for 10 h, and values are means ± SEM from at least three biological replicates. No significant growth was detected over this time frame at either initial density of 0.5 (G) or 0.005 (H).

Figure 3

Polymers enhance quorum sensing in V. cholerae. V. cholerae A1552 containing pBB1 was grown for 16 h and then adjusted to an OD600 of 0.2 in the presence of P1 (A–C), P2 (D–F), or AMW alone (G). Polymers were adjusted to final concentrations of 0.005 (A, D), 0.05 (B, E), or 0.5 (C, F) mg mL^–1 in AMW. Luminescence of samples was imaged every 30 min for 15 h, and representative images for each time point are shown from 0 h (top left) to 15 h (bottom right) of each panel. Luminescence intensities over time were analyzed by quantifying pixel intensities, and means ± SEM from at least three biological replicates are shown for P1 (left) and P2 (right panel) (H).

Figure 4

Polymer-mediated luminescence is not due to nutrient starvation within clusters. V. cholerae E7646 Δcrp containing the luminescence reporter pBB1 was grown for 16 h and then diluted to an OD₆₀₀ of 0.2 in AMW alone (black) or AMW containing P1 (A) or P2 (B) at concentrations of 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL^–1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. (C) V. cholerae E7646 wild-type containing pBB1 was grown for 16 h and adjusted to an OD₆₀₀ of 0.2 in AMW containing 1% glucose alone (orange) or in the presence of 0.5 mg mL^–1 of P1 (light blue) or P2 (dark blue). Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown.

Figure 5

Polymer-mediated enhancement of quorum sensing is mainly driven by CAI-1. V. cholerae A1552 wild-type (dark) and quorum sensing mutants containing pBB1 were grown for 16 h and then diluted into AMW to give equal cell densities and a total OD₆₀₀ of 0.2. Strains were grown together in AMW alone (black) or AMW containing P1 at 0.005 (green), 0.05 (blue), or 0.5 (red) mg mL^–1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. Mutants grown in coculture with the wild-type were (A) DH231 (ΔluxSΔcqsS) producing no AI-2, (B) WN1103 (ΔluxQΔcqsA) producing no CAI-1, (C) BH1578 (ΔluxSΔcqsA) producing no AI-2 or CAI-1, and (D) BH1651 (luxO^D47E).

Figure 6

Enhanced quorum sensing is driven by enhanced production of autoinducers in response to polymers. Cultures of V. cholerae were adjusted to an OD of 0.2 and grown for 16 h in LB medium, and the supernatants were harvested, filtered, and incubated with V. cholerae BH1578 containing pBB1. Strains used to harvest supernatants were (A) wild-type A1552, (B) DH231 (ΔluxSΔcqs), (C) WN1103 (ΔluxQΔcqsA), and (D) BH1578 ((ΔluxSΔcqsA). Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown. V. cholerae wild-type was adjusted to an OD₆₀₀ of 0.2 in AMW alone or AMW containing 0.005–0.5 mg mL^–1 P1 (E) or P2 (F), and the supernatants were harvested and filtered 16 h later. For their autoinducer content to be determined, the supernatants were incubated with the reporter strain V. cholerae BH1578 containing pBB1. Luminescence was recorded every 30 min for 10 h, and means ± SEM from at least three biological replicates are shown.

Figure 7

Polymer-mediated quorum induction overrides the canonical biofilm dissipation program in V. cholerae. V. cholerae wild-type A1552 containing pRW50T lacZ reporters for the promoters of rbmA (A) or vpsR (B) were grown for 16 h and then diluted into AMW alone or containing 0.005 or 0.05 mg mL^–1 P1 or P2 as indicated to give an OD₆₀₀ of 0.2. Following 16 h of incubation, clustered bacteria were removed and either processed for β-galactosidase assays or treated with high-salt PBS to disperse the cultures for OD₆₀₀ measurements. Transcriptional activities were calculated and normalized to untreated cultures. Shown are means ± SEM and individual measurements for three biological replicates. Statistical significance was determined by ANOVA and a Dunnett’s multiple comparison test and is depicted as ****p-values ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, and ns for not significant (p ≥ 0.05). (C) V. cholerae wild-type A1552 containing pRW50T lacZ reporter for the aphA promoter was grown in AMW alone (orange) or containing P1 (light blue) at 0.05 mg mL^–1 or P2 (dark blue) at 0.5 mg mL^–1 for 18 h. Clustered bacteria were removed at indicated times and either processed for β-galactosidase assays or treated with high-salt PBS to disperse the cultures for OD₆₀₀ measurements. Transcriptional activities were calculated and normalized to the activities of untreated cultures at 2 h. Shown are means ± SEM and individual measurements for three biological replicates.