Figure 4.

Macrophages are required to control Staphylococcus aureus infection in a (p)ppGpp-dependent manner. A, Survival of Pu.1 knockdown zebrafish larvae injected with PBS, WT, or (p)ppGpp⁰ at doses of 3000–4000 CFU at 30 hours postfertilization into the circulation valley. The Pu.1 morpholino, 1 pmol, was injected into the yolk of 1-cell stage embryos. Survival was monitored until 93 hpi when the larvae reached 5.2 days postfertilization. The experiment was performed in triplicate. Statistical significance was determined by log-rank (Mantel-Cox) test: ns between WT and (p)ppGpp⁰, P = .5275. B and C, Intracellular survival of WT S. aureus within primary human macrophages, as compared to (B) (p)ppGpp⁰ and (C) (p)ppGpp overexpression strain, Rel F128Y. MDMs were infected with bacteria at multiplicity of infection of 10 for 1 hour, before addition of 100 µg/mL gentamicin to kill extracellular bacteria. Infected MDMs were lysed at 1.5, 3, 4.5, or 6 hpi and plated to measure intracellular CFU/mL. Experiments were repeated 4 times for (B) (p)ppGpp⁰ (see also Supplementary Figure 2) and 3 times for (C) Rel F128Y (Supplementary Figure 4) using MDMs from different donors. Data from 1 representative donor are presented for each experiment. For each donor MDM population, 2 technical repeats were performed. Statistical significance was determined within each MDM donor population by unpaired t test: ns, P > .05; *P < .05; **P < .01. Abbreviations: (p)ppGpp, guanosine tetraphosphate/guanosine pentaphosphate; (p)ppGpp⁰, (p)ppGpp null mutant; CFU, colony-forming unit; hpi, hours postinfection; MDM, monocyte-derived macrophage; ns, not significant; PBS, phosphate-buffered saline; WT, wild type.