Figure 9. Survival advantage of agr priming of S.aureus absent in phagocyte NADPH-deficient murine infection.

(A) percentage of ΔagrBD (AIP-responsive in-frame deletion mutant carrying an intact RNAIII) cells and (B) bacterial burden in lung or spleen after 2 hr of intraperitoneal infection of wild-type (WT) C57BL/6 mice or phagocyte NADPH oxidase-deficient (Cybb^-/-) mice (see Figure 9—figure supplement 1 for data with other organs). ΔagrBD and ΔrnaIII mutant cultures were grown separately and mixed at a 1:1 ratio either before (primed) or after (unprimed) overnight growth, as for Figure 3. Both primed and unprimed mixtures were diluted after overnight growth, grown to early log phase (OD₆₀₀∼0.15), and used as inocula (1 × 10⁸ CFU) for intraperitoneal infection (n=2 groups of 10 mice each). After 2 hr, lungs and spleen were harvested and homogenized; aliquots were diluted and plated to enumerate viable bacteria. Output ratios and total and mutant colony forming units (CFU) from tissue homogenates were determined as for Figure 4E and H. A Mann-Whitney test (panel 9 A) or Student’s two-tailed t-test (panel 9B) were used to determine the statistical significance of the difference between primed and unprimed cultures. Error bars indicate standard deviation (**p<0.01; ****p<0.0001).

Figure 9—figure supplement 1. Long-lived protection by agr increases peritoneal fitness and dissemination to liver, kidney, and heart in both C57BL/6 mice and C57BL/6 Cybb^-/- (gp91phox/nox2) mice.

(A) Percentage of ΔagrBD or (B) colony forming units (CFU) of S. aureus RN6734 ΔrnaIII (GAW183) and ΔagrBD mutant (GAW130) cells in the indicated organ 1 hr post intraperitoneal infection of wild-type (WT) C57BL/6 mice or phagocyte NADPH oxidase deficient (Cybb^-/-) mice (see Figure 9 for data with lung and spleen). Wild-type and mutant strains were grown separately and mixed in a 1:1 ratio either before or after overnight growth, as for Figure 4. We called wild-type and mutant populations that were mixed prior to or after overnight growth; they were termed ‘primed’ and ‘unprimed,’ respectively. Both primed and unprimed mixtures were subsequently diluted, grown to early log phase (OD₆₀₀∼0.15), and used as inoculum for intraperitoneal infection with 1 × 10⁸ CFU (n=2 groups of 10 mice each). After 1 hr, the peritoneum was lavaged and the heart, kidneys, liver, lungs, and spleen (Figure 9) were harvested and homogenized. Samples were then diluted and plated to enumerate viable bacteria. Output ratios and total and mutant CFU from tissue homogenates were determined as for Figure 4E and H. A Mann-Whitney test (9 A) or Student’s two-tailed t-test (9B) were used to determine the statistical significance of the difference between primed and unprimed cultures. Error bars indicate standard deviation (**p<0.05; ****p<0.0001). Long-lived agr-mediated functions increased S. aureus pathogenesis in both wild-type and mutant mice, indicating a role for long-lived agr-mediated functions in pathogenesis other than protection from reactive oxygen species (ROS). Additionally, long-lived agr-mediated protection against ROS enhances fitness in lung and spleen (Figure 9), but it is dispensable for full virulence in other organs; protection is tissue-specific.