FIG 7.
Expression of essD does not confer any growth advantage and elicits an IL-12 response during infection. (A) Overnight cultures of wild-type USA300 and the isogenic essD::erm variant were normalized to an A600 of 5, diluted 1:100 into fresh medium, and grown with shaking at 37°C. Growth was monitored by recording the A600. (B) Aliquots of cultures shown in panel A were removed at 3, 4, and 6 h (T3, T4, and T6, respectively) and plated to assess viability. (C) S. epidermidis 12228 or S. simulans was cocultured with S. aureus USA300 or the isogenic essD::erm variant. Bacteria were mixed at a 1:1 ratio and plated on TSA. Immediately and 9 h following plating, cocultured bacteria were serially diluted and plated for enumeration of viable target cell counts. The data are presented as the average ± standard error of the mean from three independent competition experiments. (D) Identification of EssI in culture lysates of S. epidermidis (S. epi) 12228, S. simulans, and S. aureus USA300 using Western blotting. Samples were prepared and analyzed as described in the legend to Fig. 6A. (E) Cultures of P. acnes and P. granulosum grown to an A600 of 1.0 were plated on TSA, and the S. epidermidis 14.1.R1, USA300, essD::erm, or essDL546P strain was spotted on top of Propionibacterium lawns. Pictures of plates incubated at 37°C under anaerobic conditions for 72 h are shown. (F) Cohorts of C57BL/6 mice (n = 5) were inoculated intravenously with PBS (control) or with 5 × 107 CFU USA300 or with the essD::erm and essDL546P isogenic variants. Blood was sampled 12 h postinfection, and serum IL-12 (p70) was analyzed by enzyme-linked immunosorbent assay. Data were averaged, repeated for reproducibility, and analyzed by one-way ANOVA using Bonferroni's multiple-comparison test and with an unpaired t test in panel D (*, P < 0.05; ns, not significant).