FIG 2.

(A) Viscosity measurements of the exopolysaccharide shed from wild-type (RC2-wt), mutant (RC2-all2Δ), and reconstituted (RC2-all2Δ+P_ACT1-ALL2) strains grown for 14 days in minimal medium. Shed capsular polysaccharide of the all2Δ mutant was less viscous. The all2Δ mutant had increased sensitivity to H₂O₂ in vitro, as documented by incubating 10⁵ or 10⁶ RC2-wt, RC2-all2Δ, and RC2-all2Δ+P_ACT1-ALL2 cells in DMEM with 5 mM H₂O₂ for 3 h and quantification of the percent survival (B) and by spotting 5 μl on YPD agar (C). (D) Killing assays were performed by lysing murine macrophages 3 h or 6 h after coincubation and plating the CFU. all2Δ cells were more sensitive to macrophage killing, consistent with their increased sensitivity to H₂O₂ in vitro. (E) The phagocytosis index determined at 2 h after coincubation was significantly higher in macrophages incubated with RC2-all2Δ cells than in macrophages coincubated with RC2-wt or RC2-all2Δ+P_ACT1-ALL2 cells. Intracellular phagocytosis index was calculated by visually counting cells on a microscope. All experiments were performed in triplicates. An asterisk denotes a P value of ≤0.05.