Figure 6.

Deleting codY restores survival in vitro. A, Schematic representation of the indirect link between activation of the stringent response and derepression of the CodY regulon, as mediated by intracellular GTP levels. B, Susceptibility of WT, (p)ppGpp⁰, codY::Tn, and (p)ppGpp⁰  codY::Tn to 100 mM H₂O₂. Percentage bacterial survival with mean and standard deviation are plotted. Statistical analysis was performed using 1-way ANOVA with Tukey multiple comparisons test. C, Intracellular survival of WT, (p)ppGpp⁰, codY::Tn, and (p)ppGpp⁰  codY::Tn strains within primary human macrophages. MDMs were infected with bacteria at multiplicity of infection 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. Infection assays were repeated 3 times (see also Supplementary Figure 6) using MDMs from 3 different donors. Data from 1 representative donor are shown. For each donor MDM population, 2 technical repeats were performed. Statistical significance was determined within each MDM donor population by unpaired t test. D, Survival of zebrafish larvae injected with WT, (p)ppGpp⁰, codY::Tn, and (p)ppGpp⁰  codY::Tn at doses of 3000–4000 CFU at 30 hours postfertilization into the circulation. Survival was monitored until 93 hpi when the larvae reached 5.2 days postfertilization. Statistical significance was determined by log-rank (Mantel-Cox) test. The experiment was performed in triplicate. *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: (p)ppGpp⁰, guanosine tetraphosphate/guanosine pentaphosphate null mutant; CFU, colony-forming unit; CT, C-terminal; HD, hydrolase; hpi, hours postinfection; MDM, monocyte-derived macrophage; SR, stringent response; SYN, synthetase; WT, wild type.