FIG 7 .

Pyruvate enhances the virulence of USA300. (A and B) Wild-type and isogenic leucocidin-inactivated (Δluk) USA300 strains were cultured to post-exponential phase in 0% or 2% pyruvate and used to determine the impact of virulence ex vivo. (A) Primary human PMNs (2 × 10⁵ cells/well) were either incubated with filtered culture supernatants to a concentration of 0.313% to 20% with CellTiter metabolic dye or were infected with live USA300 strains at a multiplicity of infection (MOI) of 0.5 to 50 for intoxication assays measuring cell death levels. (B) Cell death was measured as the percentage of LDH release from lysed neutrophils. For both experiments, values representing "% Dead Primary PMNs” represent averages from neutrophils isolated from 4 to 5 independent donors, each incubated/infected with two USA300 colonies, ± standard errors of the means. The analyses of statistical significance were performed using a two-way ANOVA, correcting for multiple comparison using Dunnett’s analysis. Stars denote the statistical significance of results of comparisons of wild-type LAC in the presence or absence of pyruvate. Δluk = ΔhlgACB ΔlukED ΔlukSF-PVL ΔlukAB. (C) Mice (in two independent experiments conducted with a total of 10 mice per condition) were challenged via intraperitoneal infection with 0.5 × 10⁸ to 1 × 10⁸ CFU of wild-type USA300 LAC cultured to post-exponential phase in 0% or 2% pyruvate. At approximately 42 h postinfection (p.i.), mice were sacrificed and bacteria were recovered from the indicated organs to determine bacterial burden. Values representing the input or average CFU level per milliliter of bacteria per organ are shown ± standard errors of the means. The dotted line signifies the limit of detection for CFU. Analysis of statistical significance for bacterial burden was performed using a Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.