Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase–dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.

Inositol phosphates (InsP) are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here, we demonstrate that disruption of InsP6K1, one of the three mammalian InsP6Ks that convert InsP6 to InsP7, confers enhanced PtdIns(3,4,5)P3-mediated membrane translocation of Akt pleckstrin homology (PH) domain and thus augments downstream PtdIns(3,4,5)P3 signaling in murine neutrophils. Consequently, these neutrophils exhibited elevated phagocytic and bactericidal capabilities and amplified NADPH oxidase-mediated superoxide production. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. By contrast, increasing intracellular InsP7 amounts blocked chemoattractant-elicited PH domain membrane translocation and dramatically suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. These findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.