Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Abstract

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.

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

“…A number of recent S. aureus transposon-sequencing (TnSeq) studies have been carried out to identify genes that potentially contribute to fitness in vivo and/or during infection (33,34). We hypothesized that if a PPIase contributes to the folding/activity of Nuc, it is likely that the corresponding mutant would have decreased virulence/fitness in vivo.…”

“…Interestingly, mutants in each of the three PPIase genes demonstrated fitness defects under different conditions. A ppiB mutant had decreased fitness in an abscess model of infection (34), while a prsA mutant had decreased fitness in human blood and during osteomyelitis infection (33,34). A tig mutant demonstrated decreased fitness in abscess and osteomyelitis infection models (33,34).…”

Hypoxic regulation of neutrophil function and consequences for Staphylococcus aureus infection

“…aureus infection often establishes a hypoxic environment; for example staphylococcal biofilms induce hypoxia in dermal tissue, impairing wound healing [30]. A second example relates to osteomyelitis; healthy bone is intrinsically hypoxic, and further decreases in skeletal oxygen concentration upon S. aureus infection were revealed by intravital oxygen monitoring [83]. Hence, S. aureus must possess flexibility in order to survive in hypoxic environments.…”

“…Bacteria can adapt to hostile environments via two component histidine kinase systems. Transposon sequencing in a murine model of S. aureus osteomyelitis identified the staphylococcal respiratory response two component system SrrAB as essential for hypoxic survival, co-ordinating an increase in quorum sensing-dependent exotoxin production, which enhanced in vitro human osteoblast cytotoxicity [83]. Infection with an SrrA mutant decreased staphylococcal growth in vivo although, interestingly, the growth defect was rescued by depletion of neutrophils, suggesting that S. aureus requires SrrAB to resist hypoxic stress imposed by neutrophils.…”

Targeting fundamental pathways to disrupt Staphylococcus aureus survival: clinical implications of recent discoveries

“…In a variety of end-organ invasion sites, the organism must maintain virulence in the setting of reduced oxygen tension (121). A recent study used transposon sequence analysis in a robust murine model of osteomyelitis (92) to show that the two-component gene regulatory system SrrAB (122) is critical for staphylococcal survival in hypoxic bone (123). Further, the supernatant from S. aureus cultures grown under hypoxic conditions had markedly increased cytotoxicity against a variety of murine and human cells, and SrrAB appears to regulate both hypoxia-induced toxin production and quorum sensing in response to varying oxygen tension.…”