Vibrio cholerae motility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus

Abstract

The bacterial predator Bdellovibrio bacteriovorus is evolved to attack and kill other bacteria, including the human intestinal pathogen Vibrio cholerae. Although B. bacteriovorus exhibit a broad prey range, little is known about the genetic determinants of prey resistance and sensitivity. Here we perform a genetic screen on V. cholerae and identify five pathways contributing to predation susceptibility. We find that the essential virulence regulators ToxR/S increase susceptibility to predation, as mutants of these genes are more resistant to predation. We observe by flow cytometry that lipopolysaccharide is a critical defense, as mutants lacking O-antigen are rapidly attacked by predatory B. bacteriovorus. Using polymer solutions to alter media viscosity, we find that when B. bacteriovorus attacks motile V. cholerae, increased drag forces slow its ability to prey. These results provide insights into key prey resistance mechanisms, and may be useful in the application of B. bacteriovorus in treating infections.

Fig. 1

V. cholerae mutants with altered sensitivity to B. bacteriovorus predation. a We generated a complex V. cholerae transposon mutant library and subjected it to transposon-insertion sequencing (Tn-seq) before and after predation by B. bacteriovorus. Mutants with decreased fitness (w < 0.4) are shown and categorized according to gene ontology terms. b Promising transposon mutants identified in a were re-created by gene deletion and replacement with an FRT scar. This FRT scar served as a pseudo-transposon for a mini Tn-seq of a much smaller mutant library: 32 mutants compared to 50,000 in the initial Tn-seq. The color scheme of a matches b. The average fitness values and standard errors of the mean (SEM) for five biological replicates are shown. Error bars represent standard error of the mean. Significance was determined by comparing the fitness of each mutant to the average fitness values for two neutral genes. **P < 0.002; ***P < 0.0007; ****P < 0.0001 (ANOVA and Dunnett’s multiple comparisons test)

Fig. 2

V. cholerae complementation restores WT sensitivity to predation. In 1:1 competitions with WT, V. cholerae mutants showed similar sensitivity to B. bacteriovorus predation as in the previous Tn-seq experiments. Complemented strains showed WT levels of sensitivity to predation. CheY**, a tumble-biased strain, showed intermediate sensitivity to predation compared to WT and ∆motY strains. The average competitive indices and standard error of the mean for three to six biological replicates are shown. *P < 0.05 (ANOVA with Dunnett’s multiple comparisons test)

Fig. 3

V. cholerae rounding and attachment by B. bacteriovorus. a Gating strategy for analyzing B. bacteriovorus attachment to V. cholerae by flow cytometry. The left plots show all events, while right plots show green-only and double-positive events for the same experiment. b, c Predator attachment to prey by flow cytometry at MOI 1 at 30 min (b) or 60 min (c) post infection. As a control, fluorescent B. bacteriovorus and V. cholerae were fixed immediately upon mixing to account for false positive interactions. All attachment to mutant prey was normalized to WT prey attachment for three biological replicates. d Fluorescence images of GFP-expressing V. cholerae 1 h following infection with B. bacteriovorus at MOI 1. The scale bar indicates 10 µm. e The percentage of rounded V. cholerae cells was calculated by analyzing images by Matlab for roundness (eccentricity) of three biological replicates. Error bars represent standard error of the mean. Statistics were analyzed by ANOVA with Dunnett’s multiple comparison’s test

Fig. 4

Increasing viscosity reduces killing and invasion of motile V. cholerae. a, b The survival percentage of motile and non-motile V. cholerae strains in HEPES medium alone or supplemented with ficoll or methylcellulose to increase viscosity. Infections were carried out for 14 h with an MOI of 0.1. Survival percentage and SEM is shown for three to six biological replicates. c Fluorescence microscopy of B. bacteriovorus (magenta) and V. cholerae (cyan), following 1 h of infection at MOI 0.1 in HEPES or Ficoll-supplemented medium. Scale bar indicates 10 µm. d Images from c were scored for externally attached or internalized B. bacteriovorus at 30 and 60 min. The percentage of internalized attachments and SEM is shown for three biological replicates. Error bars represent standard error of the mean. Statistics were analyzed by ANOVA with Dunnett’s multiple comparisons test