Host intestinal signal-promoted biofilm dispersal induces Vibrio cholerae colonization

Abstract

Vibrio cholerae causes human infection through ingestion of contaminated food and water, leading to the devastating diarrheal disease cholera. V. cholerae forms matrix-encased aggregates, known as biofilms, in the native aquatic environment. While the formation of V. cholerae biofilms has been well studied, little is known about the dispersal from biofilms, particularly upon entry into the host. In this study, we found that the exposure of mature biofilms to physiologic levels of the bile salt taurocholate, a host signal for the virulence gene induction of V. cholerae, induces an increase in the number of detached cells with a concomitant decrease in biofilm mass. Scanning electron microscopy micrographs of biofilms exposed to taurocholate revealed an altered, perhaps degraded, appearance of the biofilm matrix. The inhibition of protein synthesis did not alter rates of detachment, suggesting that V. cholerae undergoes a passive dispersal. Cell-free media from taurocholate-exposed biofilms contains a larger amount of free polysaccharide, suggesting an abiotic degradation of biofilm matrix by taurocholate. Furthermore, we found that V. cholerae is only able to induce virulence in response to taurocholate after exit from the biofilm. Thus, we propose a model in which V. cholerae ingested as a biofilm has coopted the host-derived bile salt signal to detach from the biofilm and go on to activate virulence.

FIG 1

Effects of bile salts on V. cholerae biofilm formation. (A) Structures of select bile salts. Cholate can be dehyroxylated at the 7α position to form deoxycholate or conjugated with taurine to form taurocholate. (B) V. cholerae was inoculated into glass tubes and allowed to form biofilms for 24 h at 22°C. Biofilms of cultures grown in the presence of deoxycholate (DC), cholate (CC), glycocholate (GC), or taurocholate (TC) were quantified with crystal violet staining, and data are presented as OD₅₇₀ values. (C) Expression of vpsA during formation of WT V. cholerae biofilms in the presence of 1 mM DC and TC for 18 h, as measured by the β-galactosidase assay. Data are means and standard deviations (SD) from three independent experiments. NS, no significance; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

FIG 2

Effect of taurocholate (TC) on detachment of mature V. cholerae biofilms. (A) Remaining biofilm mass after detachment for the indicated time in the presence of 1 mM taurocholate (TC) or 1 mM deoxycholate (DC), presented as fold change of OD₅₇₀ over no bile salts added for the same incubation period. (B) Biofilm-derived planktonic cells incubated in the presence of 1 mM TC or 1 mM DC, presented as fold change of CFU/ml over samples with no bile salts added for the same incubation period. (C and D) Remaining biofilm mass (OD₅₇₀; C) and detached cells (CFU/ml; D) following 2 h of exposure to TC, DC, cholate (CC), glychocholate (GC), and 0.1% Triton X (TX). All bile salts are at 1 mM final concentration. Data are means and SD from three independent experiments. NS, no significance; ****, P < 0.0001.

FIG 3

Representative images of V. cholerae biofilms imaged using scanning electron microscopy after 24 h of growth on a 55- by 55-mm glass coverslip followed by 1 h of exposure to 1 mM TC (B, D, and F) or no TC (A, C, and E). Images are at ×250 (A and B), ×2,000 (C and D), and ×8,000 (E and F) magnification.

FIG 4

V. cholerae response to TC during detachment. (A) Level of expression of vpsA in detached cells, as measured with the β-galactosidase assay. Cells were collected following 2 h of detachment. TC and DC were supplemented to a final concentration of 1 mM. (B) Following growth for 24 h in biofilm-inducing conditions, wild-type V. cholerae biofilms were incubated with the protein synthesis inhibitor chloramphenicol (Cm) and TC for 2 h. Remaining biofilm was quantified with CV staining. Data are means and SD from three independent experiments. NS, no significance; **, P < 0.005; ****, P < 0.0001.

FIG 5

Carbohydrate content released from V. cholerae biofilms during incubation with bile salts. To reduce growth and background from rich media, biofilms were incubated in M9 minimal media with 0.2% glycerol for 2 h. Medium was filter sterilized before the carbohydrate content was estimated by the phenol-sulfuric acid method, and percent carbohydrate values were calculated by fitting to a standard glucose curve. For bile salts and taurine (TR), the final concentration is 1 mM. Data are means and SD from three independent experiments. NS, no significance; **, P < 0.005.

FIG 6

Virulence gene expression during TC-promoted biofilm detachment. (A) Expression of major virulence factor tcpA in biofilm-resident cells and detached cells as shown by a tcpA promoter-luciferase reporter fusion (P_tcpA-lux). Data are means and SD from three independent experiments. ****, P < 0.0001. RLU, relative light units. (B) Flow cytometry plot of V. cholerae harboring a plasmid with constitutive mCherry (P_tet-mCherry) and P_tcpA-gfp, in which mCherry indicates cells and GFP indicates tcpA expression. Average percentages of GFP-positive cells from three independent experiments are listed in each graph. (C) TC promotes both dispersal of individual cells from V. cholerae biofilms as well as induction of virulence program in the small intestine, synergistically enhancing colonization.