Hypermutation-induced in vivo oxidative stress resistance enhances Vibrio cholerae host adaptation
Fig 5
Biofilm formation capacity of passaged isolates and ROS resistance of biofilm-associated cells.
A. Biofilm formation. Wildtype, ΔmutS, and ΔmutS* smooth and rugose variants were cultured in LB without shaking for 16 hrs at 37°C. Culture supernatants were removed, and biofilms were washed with PBS. Biofilm formation was quantified as described previously [9]. Error bars represent means and SDs from three independent experiments. ****: One-way ANOVA test, P value <0.0001. B. Biofilm resistance to ROS. Planktonic cells (P), biofilm-associated cells (B), and disrupted biofilm cells (D) were incubated with fresh LB containing 1 mM H2O2 or 100 μM cumene hydroperoxide (CHP) for 1 hr. The surviving cells were then enumerated by serial dilution and plated onto LB agar. Error bars represent means and SDs from three independent experiments. ****: One-way ANOVA test, P value <0.0001. **: P < 0.05. C. Colonization of smooth revertants in NAC- mice. 108 cells of ΔmutS* rugose isolates and their Rif-resistant smooth revertants were mixed in a 1:1 ratio and intragastrically administered to NAC- mice. Fecal pellets were collected from each mouse at 4-day PI and plated onto selective plates. The competitive index (CI) was calculated as the ratio of smooth revertants to parental rugose colonies normalized to the input ratio. Horizontal line: mean CI of 4 mice. **: One-way ANOVA test P < 0.005 [compared with the input ratios (A5 = 1.1±0.7; B16 = 1.7±1.0; M23 = 1.3±0.7)]. D. Colonization of biofilm formation-deficiency mutants in NAC- mice. ΔmutS or ΔmutS ΔvpsA mutants were mixed with wildtype at 1:1 ratio and intragastrically administered to NAC- mice. Fecal pellets were collected from each mouse at 4-day PI and plated onto X-gal plates with appropriate antibiotics. The competitive index (CI) was calculated as the ratio of mutants to wildtype normalized to the input ratio. Horizontal line: mean CI of 4 mice. **: Mann-Whiteney test P value < 0.01.