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

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.


Introduction
Staphylococcus aureus is a major human pathogen, capable of causing a variety of life-threatening, invasive diseases and infecting nearly every organ in the human body. Yet S. aureus also innocuously colonizes the skin and nares of one-third to one-half of the population [1]. These facts suggest a remarkable flexibility in terms of metabolic and virulence programs, allowing staphylococci to adapt to diverse and changing host environments during invasive infection, while also enabling a commensal lifestyle characterized by low virulence and immunotolerance. The mechanisms by which bacterial pathogens adapt to changing host environments are poorly understood, in part due to the technical difficulty in measuring adaptive responses in vivo. However, recent advances in high-throughput sequencing have enabled an unprecedented evaluation of the host-pathogen interface. Transposon sequencing (TnSeq) is a sensitive and high-throughput tool combining highly-saturated transposon mutant libraries with massivelyparallel sequencing to calculate the fitness of all nonessential bacterial genes under a given selective pressure [2]. TnSeq has been successfully used to determine the bacterial genes required for survival in a number of different in vitro conditions and infection models [3][4][5][6][7]. More recently, a TnSeq library was generated in S. aureus and used to determine genes contributing to fitness in abscess and infection-related ecologies [8]. These studies illustrate the power of TnSeq analyses to determine the genetic requirements for bacterial adaptation to diverse host environments.
One of the most common invasive disease manifestations of staphylococcal infection is osteomyelitis, and S. aureus is by far the most common pathogen causing musculoskeletal infection. Osteomyelitis causes enormous morbidity, including functional disabilities, the requirement for invasive procedures, and the propensity to evolve into chronic infection even with appropriate management [9,10]. Two factors contribute to the therapeutic recalcitrance of osteomyelitis. First, skeletal tissues are intrinsically hypoxic, and bacterial infection further disrupts the vascular architecture of bone [11][12][13]. Second, the human skeleton is constantly being remodeled through the opposing actions of osteoblasts and osteoclasts. The kinetics of bone remodeling are affected dramatically by bacterial infection through osteo-immunologic crosstalk [14][15][16]. Thus, pathogens invading the bone must adapt to hypoxia as well as constant shifts in the available host substrates for adhesion and nutrient acquisition. While these factors would seemingly create an inhospitable environment for bacterial proliferation, bone is one of the most common locations of metastatic infection following S. aureus bacteremia [17]. One mechanism by which bacterial pathogens adapt to potentially hostile host environments is through the actions of one or more two-component systems (TCSs). Bacterial TCSs consist of a membrane bound histidine kinase sensor, which upon binding of its cognate ligand phosphorylates a response regulator. Response regulators most often function as transcriptional factors, and differentially coordinate changes in gene expression in response to a given stress. We therefore hypothesized that the ability of S. aureus to adapt to changes in available oxygen and shifts in substrate availability in inflamed skeletal tissues may rely on one or more TCSs, and that these responses would be key determinants of pathogenesis during osteomyelitis.
In this study, we employed TnSeq analysis during acute murine osteomyelitis to determine the genetic requirements for S. aureus survival during invasive infection. A large number of S. aureus genes were identified as essential for growth within bone, some of which have previously been implicated in hypoxic responses. Intravital oxygen monitoring was utilized to define changes in tissue oxygenation during osteomyelitis. Finally, we evaluated the effects of changing oxygenation on S. aureus quorum sensing and virulence factor production. Collectively, these studies determine the staphylococcal genes essential for survival during invasive infection of bone, define shifts in tissue oxygenation during invasive infection, and interrogate the mechanisms by which S. aureus can modulate its virulence in response to changes in oxygen availability.

Identification of S. aureus genes essential for invasive infection by transposon sequencing (TnSeq) analysis of experimental osteomyelitis
In order to characterize the genes required for invasive S. aureus infection, TnSeq analysis was performed during experimental osteomyelitis using a recently described S. aureus transposon insertion library in strain HG003 [8]. To identify potential bottlenecks in bacterial survival during osteomyelitis, a timecourse infection was first performed by inoculating murine femurs with strain HG003. An inoculum of 5x10 6 CFU was chosen based on direct comparison with strain LAC, which has served as the wildtype strain in prior osteomyelitis analyses and is representative of the most common lineage (USA300) of strains causing osteomyelitis in the United States [18]. At days 1, 3, 5, 7, and 12 post-infection, infected femurs were harvested and processed for CFU enumeration. After an initial period of replication from day 1 to day 3 postinfection, decreases in bacterial burdens were noted by days 5 and 12 (S1 Fig). Day 5 was therefore chosen for TnSeq analysis of acute osteomyelitis, as it likely represents the first bottleneck encountered by invading bacteria. For TnSeq analysis of osteomyelitis, mice were infected with the TnSeq library by direct inoculation into the femur. Five days post-infection, femurs from infected mice were processed for genomic DNA extraction. One limitation of TnSeq analysis during invasive infection is the requirement for an outgrowth step after the recovery of bacteria from infected tissues. Although in vitro outgrowth could potentially confound fitness calculations, it is necessary to decrease host DNA contamination and allow for efficient sequencing of microbial DNA, and thus has become a standard practice during TnSeq analysis of invasive infection models [5,8,[19][20][21][22][23]. We opted for a short outgrowth in liquid media to minimize any confounding effects on fitness calculation. For an in vitro comparator, an equivalent volume of the osteomyelitis inoculum was grown in vitro for 24 hours prior to collection and genomic DNA extraction. To determine mutants with compromised in vivo fitness, a "dval" was calculated for each gene in each condition (inoculum, in vitro comparator, or osteomyelitis).
A total of 65 genes were found to be essential for survival during osteomyelitis (S1 Table) but not in vitro growth, and mutations in an additional 148 genes resulted in significant in vivo compromise relative to the in vitro comparator (S2 Table). Of the 213 genes identified by TnSeq, 39 essential and 73 compromised genes encode hypothetical proteins, respectively. Of the remaining 101 genes, 12 essential genes and 32 compromised genes have Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers. Thirty-two of the 44 genes with KEGG identifiers can broadly be categorized into metabolic pathways, with specific pathways represented including carbon metabolism (9 genes), amino acid biosynthesis (7 genes), and the TCA cycle (5 genes). In the TCA cycle, mutations in genes sucB (SAOUHSC_01416), sucC (SAOUHSC_01216), and sucD (SAOUHSC_01218), which encode enzymes responsible for the conversion of α-ketoglutarate to succinate, each resulted in compromised growth during osteomyelitis. Moreover, genes encoding enzymes in pathways that feed into the TCA cycle were also important for intraosseous growth, including pyruvate carboxylase (SAOUHSC_01064 pyc), pyruvate dehydrogenase (SAOUHSC_01040 pdhA), and a putative malic enzyme (SAOUHSC_01810). Mutations in 7 S. aureus genes encoding amino acid biosynthesis enzymes compromised bacterial growth during osteomyelitis, yet did not significantly impair growth in vitro. These genes encode enzymes in the biosynthetic pathways for tryptophan (SAOUHSC_01369 trpC, SAOUHSC_01367 trpG, and SAOUHSC_01377), cysteine (SAOUHSC_00488 cysK), lysine (SAOUHSC_01868), leucine (SAOUHSC_02288 leuD), and the conversion of serine to glycine (SAOUHSC_02354 glyA). Mutations in 6 genes encoding components of purine and pyrimidine metabolic pathways resulted in significant in vivo compromise during osteomyelitis. Two of these genes (SAOUHSC_02126 purB, SAOUHSC_02360 tdk) were essential for staphylococcal survival in bone. A substantial portion of the oxidative phosphorylation pathway was also found to be necessary for staphylococcal growth during osteomyelitis. Four of the 12 essential genes with KEGG identifiers and 1 of the mutants with compromised growth are involved in oxidative phosphorylation, including components of quinol oxidase complexes (SAOUHSC_01000 qoxC, SAOUHSC_01032 cydB), and 3 subunits of the F-type ATPase (SAOUHSC_02340 atpC, SAOUHSC_02343 atpG, SAOUHSC_02346 atpH). Collectively, the results of TnSeq analysis during experimental osteomyelitis suggest broad adaptations in metabolism and energy production are required for staphylococcal survival during invasive infection of bone.
In contrast to an abundance of genes encoding hypothetical proteins or metabolic pathways, relatively few genes encoding known or putative virulence factors were identified by TnSeq as important for staphylococcal survival in bone. Phosphatidylglycerol lysyltransferase, encoded by mprF (SAOUHSC_01359), catalyzes the modification of phosphatidylglycerol with L-lysine and contributes to bacterial defenses against neutrophils, cationic antimicrobial peptides, and certain antibiotics [24]. The mprF gene was essential for growth during osteomyelitis, suggesting that resistance to antimicrobial peptides and neutrophils are important components of staphylococcal survival in bone. A second virulence-associated gene identified by TnSeq as essential for S. aureus osteomyelitis was isdF (SAOUHSC_01087), which encodes a component of the iron-regulated surface determinant heme uptake system [25]. Interestingly, mutation of the ferric uptake regulator (SAOUHSC_00615 fur) gene also resulted in compromised intraosseous growth, illustrating a potential role for iron acquisition in the pathogenesis of staphylococcal osteomyelitis. Mutation in the genes encoding thermonuclease (SAOUHSC_00818 nuc), a fibrinogen-binding protein (SAOUHSC_01110), the repressor of toxins (SAOUHSC_01879 rot), and two serine proteases (SAOUHSC_01935 splF, SAOUHSC_01938 splD) also compromised the survival of S. aureus during osteomyelitis.
The genes identified by TnSeq as critical for staphylococcal osteomyelitis encode diverse metabolic processes, hypothetical proteins, and select virulence factors. These results suggest that complex bacterial adaptations occur in response to invasive infection of bone. One mechanism by which bacterial pathogens sense and ultimately respond to host-imposed stresses is through TCSs. We therefore hypothesized that staphylococcal TCSs might coordinate the complex adaptations observed during osteomyelitis. Strikingly, TnSeq analysis identified only one S. aureus TCS as required for intraosseous survival. The staphylococcal respiratory response (SrrAB) system is involved in coordination of the staphylococcal response to hypoxia and other stresses [26], and has been shown to directly regulate select virulence factors [27]. Both the histidine kinase (srrB) and the response regulator (srrA) components of the SrrAB locus were essential for staphylococcal survival in bone, implying that this TCS might be particularly important for coordination of the metabolic and virulence adaptations to intraosseous growth (S1 Table). In total, these results reveal the power of TnSeq analysis to identify S. aureus genes required for invasive infection of bone.
SrrAB differentially regulates S. aureus genes under aerobic and hypoxic growth, and is required for survival during osteomyelitis Among the mutants that exhibited decreased survival in the osteomyelitis model, we identified a single TCS, SrrAB, which coordinates responses to hypoxia and nitrosative stress in vitro [26]. Moreover, mutations in two additional genes regulated by SrrAB specifically under conditions of nitrosative stress, cydB and qoxC, also resulted in significantly decreased fitness during osteomyelitis (S1 and S2 Tables) [26]. Bone and bone marrow are intrinsically hypoxic, leading to the hypothesis that SrrAB contributes to osteomyelitis pathogenesis by sensing and responding to changes in environmental oxygen [11,28]. Because the SrrAB regulon was previously defined under conditions of nitrosative stress, we sought to further define the oxygendependent SrrAB regulon by performing global transcriptional analysis of a clinically relevant strain (LAC) in comparison to a mutant strain lacking srrAB expression (ΔsrrA) in both aerobic and hypoxic growth conditions. Inactivation of srrAB under aerobic conditions resulted in the differential regulation of 64 genes (39 transcripts increased in abundance and 25 decreased in abundance upon inactivation of srrAB) (S3 Table). Under hypoxic growth conditions, srrAB inactivation led to differential regulation of 78 genes (22 transcripts increased in abundance and 56 decreased in abundance) (S4 Table). Of the genes differentially regulated by SrrAB under aerobic or hypoxic conditions, only 16 were previously identified as members of the SrrAB regulon under nitrosative stress, suggesting that specific stresses might invoke different SrrAB-dependent transcriptional responses [26]. Moreover, by defining the SrrAB regulon under aerobic versus hypoxic conditions, we discovered that an additional 7 genes important for survival during osteomyelitis in the TnSeq dataset are also SrrAB-regulated (S3 and S4 Tables). The requirement of multiple genes in the SrrAB regulon for survival during osteomyelitis suggests that the SrrAB TCS is an important orchestrator of S. aureus stress responses in inflamed skeletal tissues.
Previous reports have demonstrated a significant defect in the growth of an srrAB mutant under anaerobic conditions but not under hypoxic growth conditions [26,27,29]. To confirm that SrrAB was not found to be essential in the TnSeq analysis simply because of a defect in growth, the ΔsrrA polar transposon mutant and mutations in known genes of the SrrAB regulon (pflA, pflB, qoxA, and qoxC) were analyzed in the LAC strain background. The growth rate of each mutant under aerobic or hypoxic conditions was monitored over time. Under aerobic and hypoxic growth conditions, ΔsrrA had an enhanced lag phase compared to WT but reached equivalent optical densities to WT by 8 hours (S2 Fig). The ΔqoxA and ΔqoxC mutants were unable to reach maximal optical densities as previously reported due to disruption of the electron transport chain [26]. These results indicate that an srrAB mutant is not impaired for growth under hypoxia, further validating our TnSeq methods.
To investigate the role of SrrAB in osteomyelitis in a clinically-relevant background without the potentially confounding influence of competition from other mutants in the TnSeq library, groups of mice were infected with either WT or ΔsrrA in the LAC background. At 5 or 14 days post-infection, femurs were either processed to quantify bacterial burdens or subjected to micro-computed tomography (microCT) imaging (day 14) for quantification of cortical bone destruction. Inactivation of SrrAB resulted in a significant reduction in bacterial burdens in infected femurs at both 5 and 14 days post-infection ( Fig 1A). Moreover, murine femurs infected with ΔsrrA sustain significantly less cortical bone destruction than WT-infected femurs ( Fig 1B-1D). These results demonstrate that SrrAB is critical for S. aureus survival in infected bone and for induction of pathologic changes in bone remodeling during osteomyelitis. Furthermore, the data suggest that staphylococci encounter hypoxic and/or nitrosative stresses during osteomyelitis.

S. aureus osteomyelitis triggers reduced oxygen availability in skeletal tissues
Normal bone and bone marrow are intrinsically hypoxic, with a physiologic oxygen concentration range of 11.7 to 48.9 mmHg (1.5-6.4% O 2 ), compared to atmospheric oxygen at approximately 160 mmHg (21% O 2 ) [11,28]. TnSeq analysis demonstrated that the hypoxiaresponsive SrrAB TCS is essential for S. aureus survival in bone, suggesting that bacterial invasion and the resulting inflammation associated with osteomyelitis trigger further decreases in skeletal oxygen availability. In order to determine the oxygen concentrations of S. aureus infected murine femurs during osteomyelitis, an Oxylite monitor was used to record oxygen tensions at the inoculation site at various times post-infection. In uninfected mice, average pO 2 in the intramedullary canal was 45.2 mmHg, (Fig 2) consistent with previously reported bone marrow physoxia [28]. As infection progressed, the infectious focus became increasingly hypoxic, with an average oxygen tension of 14.3 mmHg at 10 days post-infection. This decreased oxygen tension was not due to the trauma induced by the inoculation procedure, as mockinfected bone showed an elevated mean pO 2 of 77.5 mmHg by 4 days post-procedure (Fig 2). Collectively, these findings demonstrate that skeletal tissues become increasingly hypoxic during S. aureus osteomyelitis.
The srrAB promoter is active in hypoxic skeletal tissues Intravital pO 2 monitoring revealed that skeletal tissues become increasingly hypoxic during osteomyelitis, with dramatically reduced oxygen tensions as early as 24 hours after infection. These data and the results of TnSeq analysis suggest that the srrAB promoter is active in vivo.
To test the hypothesis that srrAB promoter activity increases with decreasing oxygen availability in infected skeletal tissues, a luminescent reporter construct was created in which expression of the luxABCDE operon is driven by the srrAB promoter. Mice were infected with WT S. aureus containing either this construct or a promoterless vector control, and at 1 hour or 24 hours post-infection infected femurs were explanted and immediately imaged for bioluminescence. No detectable luminescence above background was detected in femurs infected with WT bacteria containing the promoterless control plasmid at 1 hour or 24 hours after infection (Fig 3). In contrast, femurs infected with the PsrrAB-pAmiLux construct showed no detectable luminescence above background at 1 hour post-infection, but displayed strong luminescent signal at 24 hours after infection, corresponding to the induction of hypoxia in infected skeletal tissues (Fig 3). Collectively, these results demonstrate that the srrAB promoter is activated in vivo during infection of hypoxic skeletal tissues.

Neutrophil depletion rescues the intraosseous growth defect of an srr mutant
Intravital oxygen monitoring revealed hypoxia of skeletal tissues upon infection with S. aureus, suggesting that inflammation triggers a reduction in skeletal oxygen concentrations. Neutrophils are a significant source of both oxidative and nitrosative stresses in vivo and contribute to formation of oxygen-limited abscesses in response to staphylococcal infection [30]. To test the hypothesis that SrrAB is necessary to resist hypoxic and/or nitrosative stresses imposed by  neutrophils in vivo, mice were either rendered neutropenic with serial anti-Ly6G (1A8) monoclonal antibody injections or given an isotype control monoclonal antibody and subsequently infected with WT or ΔsrrA [31]. At 14 days post-infection, femurs were processed for enumeration of bacterial burdens. In mice treated with control antibody, a significant virulence defect was again observed in mice infected with the ΔsrrA mutant (Fig 4). However, in mice administered the anti-Ly6G (1A8) antibody, a significant increase in bacterial burdens was observed upon infection with ΔsrrA, such that bacterial burdens no longer differed significantly from non-neutropenic mice infected with WT (Fig 4). These results suggest that intraosseous survival requires SrrAB to resist hypoxic and/or nitrosative stresses produced by neutrophils in response to S. aureus osteomyelitis. S. aureus modulates quorum sensing and exotoxin production in response to oxygenation The observation that S. aureus infection of murine skeletal tissues leads to dramatically reduced oxygen concentrations prompted further evaluations of how oxygenation impacts the production of staphylococcal virulence factors. We previously demonstrated that secreted toxins regulated by the accessory gene regulator (agr) locus are particularly important for the pathogenesis of S. aureus osteomyelitis [32,33]. The agr locus (agrABCD) encodes a quorum sensing system coupled to a TCS, and is responsible for growth phase-dependent regulation of a number of S. aureus virulence factors [34]. The response regulator of the agr locus, AgrA, directly regulates the production of alpha-type phenol soluble modulins (PSMs), which contribute significantly to the pathology of S. aureus osteomyelitis [32,35]. Indeed, alpha-type PSMs were found to be the sole mediators of cytotoxicity in concentrated culture supernatant towards murine and human osteoblasts in vitro [32]. However, a recent report demonstrated that alpha-type PSM expression is directly linked to alpha toxin (Hla) expression [36]. To verify that PSMs are the sole mediators of cytotoxicity toward osteoblastic cells in S. aureus concentrated supernatants, strain LACΔpsmα1-4 (Δpsm) containing the overexpression vector pOS1-plgt driving hla expression in trans was tested for cytotoxicity towards osteoblastic cells (S3 Fig). While WT supernatant displayed maximum cytotoxicity, Δpsm and Δpsm pOS1-plgt-hla did not show significantly different cytotoxicity from control. Deletion of hla in an erythromycin-resistant LAC background also failed to attenuate cytotoxicity (S3 Fig). Moreover, targeted inactivation of RNAIII in LAC did not decrease cytotoxicity, further supporting the AgrA-regulated alphatype PSMs as the sole secreted mediators of cytotoxicity toward osteoblastic cells (S4 Fig).
To determine the impact of culture oxygenation on S. aureus exotoxin production, concentrated supernatants were prepared from S. aureus grown either aerobically or under limited oxygenation. Incubation of several different mammalian cell lines or primary human osteoblasts with varying amounts of concentrated culture supernatant demonstrated dose- dependent cytotoxicity that significantly increased if the bacteria were cultured under lower oxygenation (Fig 5). This phenomenon was not strain dependent, as hypoxic growth also increased the cytotoxicity of strains MW2 and Newman towards osteoblastic cells (S5 Fig). These results indicate that S. aureus virulence factor production is modulated in response to environmental oxygen levels.
SrrAB regulates select virulence factors under microaerobic conditions in part by directly interacting with the agr P2 and P3 promoters [27,29]. This observation, combined with the role of SrrAB in responding to hypoxic stresses led to the hypothesis that SrrAB may regulate quorum sensing and virulence factor production in response to changes in oxygenation. To investigate the impact of SrrAB on PSM-mediated killing of osteoblasts, osteoblastic cells were incubated with varying amounts of culture supernatant from WT or ΔsrrA strains grown either aerobically or under hypoxia. Aerobically grown ΔsrrA supernatants demonstrated dosedependent killing of murine osteoblasts that was significantly increased compared to aerobically grown WT supernatants, mimicking the effect of hypoxia on cytotoxicity (Fig 6A). The cytotoxicity of aerobically grown ΔsrrA was diminished by expression of the srrAB locus in trans (S6 Fig). These data suggest that SrrAB represses PSM-mediated cytotoxicity under aerobic conditions.
Because SrrAB repressed PSM-mediated cytotoxicity under aerobic conditions, we hypothesized that SrrAB impacts quorum sensing in response to oxygenation. To test this hypothesis, the reporter plasmid pDB59 (agrP3 promoter driving YFP expression) was introduced into WT and ΔsrrA [37]. Aerobically grown WT cultures demonstrated significantly lower agrP3 activation compared to cultures grown under limited oxygenation (Fig 6B). This decrease was partially SrrAB dependent, as aerobically grown ΔsrrA strains demonstrate a 2-fold higher expression of agrP3 than aerobic WT cultures (Fig 6B). To further confirm these results, quantitative RT-PCR was conducted on aerobically and hypoxically grown cultures of WT and ΔsrrA. Transcription of agrA was increased relative to aerobically grown WT for both ΔsrrAB and hypoxically grown cultures. Hypoxically grown cultures also demonstrated significantly elevated levels of psmα and RNAIII transcripts (Fig 6C). Inactivation of SrrAB resulted in an over 30-fold increase in psmα1-4 transcription and a near 20-fold increase in RNAIII expression under aerobic conditions. Under hypoxic conditions, inactivation of srrAB resulted in a 3000-fold and 160-fold increase in psmα1-4 and RNAIII transcript levels, respectively. Collectively, these data indicate that S. aureus quorum sensing and resultant cytotoxicity towards mammalian cells is modulated in an SrrAB-dependent manner in response to changing oxygen availability, and further define SrrAB as an important regulator of metabolic and virulence adaptations during invasive infection.

Discussion
TnSeq analysis during experimental osteomyelitis revealed S. aureus genes essential for invasive infection. Among the mutants with reduced in vivo fitness was one TCS, SrrAB, which was previously characterized as a coordinator of hypoxic and nitrosative stress responses [26]. SrrAB was originally identified as a regulator of oxygen-dependent toxic shock syndrome toxin-1 (TSST-1) expression, and was noted to have homology to the global respiratory regulator ResDE in Bacillus subtilis [29,38]. Subsequent analyses revealed that SrrA is capable of binding to the agr P2 and P3 promoter regions, and that overexpression of SrrAB reduces virulence in a rabbit endocarditis model [27]. These findings indicate a link between SrrAB and quorum sensing and suggest that oxygenation could impact staphylococcal virulence. Yet the specific signal(s) that activate SrrAB, and the mechanism by which this system modulates quorum sensing have yet to be determined.
Our data suggest that aerobic growth of S. aureus limits quorum sensing and agr-dependent virulence factor production in a manner that is partially dependent on SrrAB. Conversely, hypoxic growth results in significantly increased cytotoxicity toward mammalian cells. Since equivalent bacterial densities were achieved under conditions of hypoxic and aerobic growth, these results imply that the output of quorum sensing can be functionally uncoupled from bacterial density by changes in culture oxygenation. Such uncoupling could be particularly advantageous for quenching of virulence factor production in environments with higher oxygen availability, such as during colonization of the skin or nares. Since inactivation of SrrAB under aerobic conditions failed to fully restore quorum sensing and cytotoxicity to the levels observed with hypoxic growth, it is likely that this phenomenon is a result of multiple factors. Additional studies are therefore needed to determine the SrrAB-dependent and SrrAB-independent mechanisms by which oxygenation regulates quorum sensing. To this end, it has previously been demonstrated that both the S. aureus autoinducing peptide (AIP) and AgrA can be functionally inactivated by oxidation [39,40], suggesting a potential SrrAB-independent mechanism for modulation of quorum sensing by oxygen. Moreover, the S. aureus genome is known to encode other redox-sensitive regulators such as Rex, MgrA, SarA, and AirSR [41][42][43][44][45]. It is therefore possible that environmental oxygen is not a direct regulator of quorum sensing, but rather that a change in the redox status of the bacterial cell or oxidative damage triggers changes in virulence factor production. Nevertheless, our findings suggest that shifts in available oxygen, as well as the inherent differences in physiologic oxygen concentrations in various host tissues, could have a significant impact on staphylococcal virulence. Additionally, these data highlight the importance of in vitro culture conditions on the study of staphylococcal virulence.
Global transcriptional analyses defined the SrrAB regulon of S. aureus under conditions of aerobic and hypoxic growth. Interestingly, although some overlap was noted with the previously reported SrrAB nitrosative stress regulon, we identified additional SrrAB-regulated genes under conditions of changing oxygenation [26]. Although these findings may relate to technical issues or strain-dependent differences in gene regulation, they suggest that SrrAB may integrate multiple environmental signals, or that oxidative and nitrosative stress trigger a common endogenous bacterial pathway that activates SrrAB. In order to begin defining the host components that trigger hypoxic or nitrosative stress responses in S. aureus, we examined the role of neutrophils during osteomyelitis. Neutrophils impose nitrosative and oxidative stress to invading pathogens through the respiratory burst, which generates reactive oxygen and nitrogen species. Moreover, neutrophils contribute to tissue hypoxia through abscess formation [30]. In support of the role of SrrAB in resisting such hypoxic and nitrosative stresses, we found that neutrophil depletion partially rescued the virulence defect of an srrA mutant. Additional studies are needed to parse out the effects of neutrophil-derived reactive oxygen and nitrogen species versus abscess-associated tissue hypoxia on the survival of S. aureus. Furthermore, it is likely that other innate and adaptive immune responses contribute to changes in tissue oxygenation and thus the redox status of invading pathogens. In addition to the genes encoding SrrAB and its targets, TnSeq analysis during osteomyelitis revealed a large number of staphylococcal mutants with compromised fitness in vivo. Many of the genes identified as essential or compromised during in vivo growth can be broadly classified as associated with metabolism. In contrast, very few prototypical virulence factors were identified as essential for osteomyelitis. The lack of traditional secreted virulence factors identified through TnSeq analysis is not surprising due to the nature of the technique. Infection with a pooled transposon library allows for mutants deficient in a particular gene to potentially co- S. aureus modulates quorum sensing and exotoxin production in response to oxygenation. Supernatants from WT or ΔsrrA were prepared by inoculating RPMI and 1% CA with a 1:1000 dilution from overnight cultures and growing for 15 hours either aerobically or hypoxically. Identical culture conditions were used to monitor quorum sensing and transcript levels (see below). (A) MC3T3 cells were seeded into 96 well plates at 5,000 cells per well. After 24 hours, growth media was replaced, and 20% or 30% of the total media volume was replaced with concentrated culture supernatant grown either aerobically or hypoxically, or an equivalent volume of RPMI. Cell viability was assessed 24 hours later, and results are expressed as percent of RPMI control (n = 10). Results are representative of at least three independent experiments. Error bars represent the SEM. (B) Agr-mediated quorum sensing was monitored using agrP3-dependent YFP expression in WT or ΔsrrA strains grown aerobically or hypoxically as above. YFP relative fluorescent units (RFUs) were averaged from 3 technical replicates. Error bars represent the SD. Data shown are an average of 3 biologically independent experiments. RFUs monitored at 0, 6, 9, 12, and 15 hours after back-dilution from overnight culture. * and ** represent p<0.05 and 0.01, respectively relative to WT aerobic at 15 hours as determined by Student's t test. # represents p<0.05 relative to ΔsrrA aerobic. (C) cDNA samples from WT or ΔsrrA strains grown aerobically or hypoxically as above for 15 hours were subjected to qRT-PCR. Graph depicts fold change of the indicated transcripts relative to WT aerobic transcript level. Data shown are an average of 3 biologically independent experiments. Error bars represent the SEM. Significance was determined by two way ANOVA. * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001 relative to WT aerobic. opt bacterial factors from other mutants. Indeed, this phenomenon has been characterized for the exchange of metabolic intermediates in S. aureus, and it is conceivable that secreted virulence factors could be "shared" in a similar manner [33]. An additional limitation of TnSeq analysis is the requirement for a short outgrowth step in liquid media following harvest of the transposon library from infected bone. This outgrowth step could potentially confound our results by altering the fitness of mutants recovered from infected tissues. However, a brief outgrowth step is necessary to decrease the amount of murine DNA present in the femur homogenate and allow for effective sequencing of bacterial DNA, and is a common adjustment in TnSeq analyses of infected tissues [5,8,22].
TnSeq analysis of staphylococcal osteomyelitis paralleled a previous TnSeq analysis of staphylococcal growth in soft tissue abscesses [8]. In fact, 40 of the 65 genes identified as essential for growth during osteomyelitis were also essential for growth in murine abscesses. This observation is consistent with our previous data showing that osteomyelitis is characterized by exuberant abscess formation in the bone marrow [32], and suggests common stresses are encountered by staphylococci in neutrophil-rich inflammatory lesions. However, 25 of the 65 genes essential for intraosseous survival were not found to be essential for abscess growth, and may reflect unique adaptations to colonization of skeletal tissues.
Of the genes required for S. aureus survival during invasive infection, many encode hypothetical proteins or proteins without a previously characterized role in virulence. This observation highlights the power of TnSeq analysis as an unbiased evaluation of the genetic requirements for bacterial survival in host tissues. In summary, the results of this study elucidate bacterial survival strategies during invasive infection, link changes in environmental oxygen to staphylococcal quorum sensing and virulence, and provide a firm foundation to identify new targets for antimicrobial and vaccine design.

Ethics statement
All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Vanderbilt University and performed according to NIH guidelines, the Animal Welfare Act, and US Federal law.

Bacterial strains and growth conditions
The S. aureus TnSeq library in strain HG003 has been previously described [8]. All other experiments were conducted in an erythromycin-sensitive, tetracycline-sensitive derivative of the USA300 strain LAC (AH1263), which served as the wildtype (WT) unless otherwise noted [46]. Strain LACΔpsmα1-4 has been previously described [32,47]. Strains ΔsrrA, ΔqoxA, ΔqoxC, ΔpflA, and ΔpflB in the LAC background were created by bacteriophage phi-85-mediated transduction of erm-disrupted alleles from the respective JE2 strain mutants obtained from the NARSA transposon library [48]. Strains Δpsm pOS1-plgt and Δpsm pOS1-plgt-hla were provided by Dr. Juliane Bubeck-Wardenburg [36]. Construction of strain LAC ΔRNAIII is described below. Plasmid pDB59 (agr-P3-YFP) was electroporated into LAC or ΔsrrA for monitoring of agr-dependent quorum sensing [37]. All strains were grown in glass Erlenmeyer flasks at 37°C with orbital shaking at 180 rpm. All S. aureus strains were grown in Tryptic Soy Broth (TSB), Brain-Heart Infusion (BHI), or Roswell Park Memorial Institute medium (RPMI) supplemented with 1% casamino acids (CA). Escherichia coli was grown in Luria Broth (LB). Erythromycin and chloramphenicol were added to cultures at 10 μg ml -1 where indicated. Ampicillin was added to cultures at 100 μg ml -1 where indicated. Cadmium chloride was added to cultures at 0.1 mM where indicated. A 5:1 flask to volume ratio was utilized unless otherwise noted. For comparative growth analyses, overnight aerobic cultures were back-diluted 1:1000 into fresh TSB or BHI media and optical density at 600 nm (OD 600 ) was monitored over time.

Construction of LAC ΔRNAIII
RNAIII including upstream and downstream flanking regions were amplified using primers 5'-GCATGCGTCGATATCGTAGCTGGGTCAG-3' and 5'-GAATTCGAAGTCACAAGTAC TATAAGCTGCG-3', and cloned into the HincII site of pUC18 [49] to create pGAW1. To delete RNAIII, inverse PCR was performed with primers 5'-TTTGGGCCCTATATTAAAAC ATGCTAAAAG-3' and 5'-TTTCTCGAGGTAATGAAGAAGGGATGAGTT-3' amplifying RNAIII flanking regions and the remaining plasmid backbone of pGAW1. The vector was religated after treatment with Polynucleotide Kinase (New England Biolabs, MA) and designated pGAW3. To insert an antibiotic resistance cassette, pGAW3 was digested with ApaI and XhoI, religated with the ApaI-XhoI fragment from pJC1075 [50] (cadCA, conferring resistance to cadmium) and designated pGAW6. The SphI-KpnI fragment from pGAW6 was cloned into the allelic replacement vector pJC1202 [50] using the same restriction sites and designated pGAW7. Strain RN4220 was electroporated with plasmid pGAW7 and plated on GL agar containing 5 μg chloramphenicol ml -1 at 30°C. Allelic exchange was carried out as previously described [50]. Phage 80a was then used to transduce the mutation into LAC to generate LAC RNAIII::cad, herein designated LAC ΔRNAIII.

Construction of an SrrAB overexpression plasmid
To express srrAB in trans, the srrAB open reading frame was PCR amplified from genomic DNA of LAC using primers 5'-ATCTCGAGATGTCGAACGAAATACTTATCG-3' and 5'-ATGGATCCTTCAATTTTATTCTGGTTTTGGTAG-3'. The resulting srrAB amplicon was then cloned into the shuttle vector pOS1 under control of the lgt promoter [51]. As a control, wild type and ΔsrrA strain LAC were transformed with pOS1-lgt lacking an insert.

Construction of an srrAB bioluminescent reporter
To examine expression of srrAB in vivo the srrAB promoter was PCR amplified from genomic DNA of LAC using primers 5'-TACCCGGGTGTATTTATCACAAAGTTTGAGAAT-3' and 5'-ATCGTCGACACAGGTCATACCTCCCAC-3'. The resulting amplicon was then cloned into the shuttle vector pAmiLux, kindly provided by Dr. Julian Davies [52]. As a control, wild type stain LAC was transformed with pAmilux lacking an insert.

Murine model of osteomyelitis and micro-computed tomographic analysis
Osteomyelitis was induced in 7-to 8-week old female C57BL/6J mice as previously reported [32]. An inoculum of 1x10 6 colony-forming units (CFU) in 2 μl PBS was delivered into murine femurs. For some experiments, mice were rendered neutropenic by serial intraperitoneal injections of an anti-Ly6G (clone 1A8) monoclonal antibody (BioXcell, West Lebanon, NH) at days -3, 0, 4, 7, and 10 post-infection. As a control, mice received serial injections of an isotype control antibody (rat IgG2a). At various times post-infection, mice were euthanized and the infected femur was removed and either processed for CFU enumeration or imaged by microcomputed tomography (microCT). For CFU enumeration, femurs were homogenized and plated at limiting dilution on Tryptic Soy Agar (TSA). Analysis of cortical bone destruction was determined by microCT imaging as previously described [32]. Differences in cortical bone destruction and bacterial burdens were analyzed using Student's t test.

Bioluminescent imaging
Bioluminescent imaging was performed on infected femurs explanted into sterile multiwell plates at either 1 or 24 hours after infection with WT bacteria containing PsrrAB-pAmiLux or pAmiLux. Luminescence was measured in an IVIS 200 Imaging System (Perkin Elmer, Akron, OH) with an exposure time of 5 minutes, f-stop of 1, and binning of 4. All images were manually scaled to the same minimum and maximum values to exclude background and include the peak luminescent value.

Intravital measurements of oxygen concentration
Intravital oxygen concentrations were measured in infected femurs using an Oxylite (Oxford Optronix, United Kingdom) oxygen and temperature monitor in conjunction with a flexible bare-fibre sensor. Mice were anesthetized with isoflurane and the surgical incision was reopened. Oxygen readings were obtained by insertion of the sensor directly through the intramedullary canal and into the infectious focus. Measurements from the probe were recorded at least 5 minutes after probe placement to allow for temperature equilibration and stabilization of oxygen readings.

Transposon sequencing analysis of experimental acute osteomyelitis
The S. aureus TnSeq library in the HG003 background has been previously described [8]. In order to identify potential bottlenecks in the murine osteomyelitis model that could confound TnSeq analysis, groups of mice were first infected with strain HG003 using an inoculum of 5x10 6 CFU and then at various times post-infection the infected femurs were collected and processed for CFU enumeration. Day 5 was chosen as a timepoint for TnSeq analysis of acute osteomyelitis as it likely represents the first bottleneck encountered by invading bacteria. To prepare the TnSeq library for inoculation into murine femurs, an aliquot of the library containing 5x10 7 CFU/ml was thawed and inoculated into 100 ml of BHI media in a 500 ml Erlenmeyer flask. This culture was incubated at 37°C for 12 hours and then back-diluted 1:100 into fresh BHI at the same flask to volume ratio and grown an additional 3 hours. Bacterial cells were harvested by centrifugation and resuspended in PBS to a concentration of 7x10 6 CFU in 2 μl PBS. This inoculum dose failed to cause mortality or severe morbidity requiring euthanasia when administered to five wildtype mice by retro-orbital injection (mice were monitored for a total of 4 days). Genomic DNA was prepared from the inoculum using a Qiagen DNeasy Kit with 40 μg ml -1 lysostaphin added to the lysis buffer. The inoculum was used to initiate experimental osteomyelitis in groups of mice as above. Another equivalent aliquot of the inoculum was seeded into a 50 ml BHI culture in a 250 ml Erlenmeyer flask. This culture was grown for 24 hours, after which time the bacterial cells were harvested and genomic DNA was prepared as above. This genomic DNA served as the in vitro comparator for TnSeq analysis. At 5 days post-infection, mice inoculated with the TnSeq library were euthanized, and the infected femurs were harvested and homogenized in 1 ml of PBS. 500 μl of this homogenate was archived by freezing at -80°C in 20% glycerol and the remaining 500 μl of the homogenate was seeded into 4ml of BHI media and cultured at 37°C and 180 rpm shaking for 5.5 hours. Bacteria were then collected by centrifugation and subjected to genomic DNA preparation as above. Recovered bacteria from 2 mice were pooled, and 3 biologically independent groups of mice were analyzed separately. Genomic DNA samples were subsequently prepared for sequencing on an Illumina HiSeq 2000 (Tufts University Genomic Core Facility). Sequencing, data analysis, and fitness calculations were performed as previously reported [8]. Briefly, a "dval" was calculated for each gene in each condition (inoculum, in vitro comparator, or osteomyelitis). The dval represents the observed number of mappable reads of insertions in a gene, divided by the number of mappable reads of insertions predicted for that gene based on its size relative to the genome and the total number of mappable reads obtained for that experiment. Genes with dval of 0.01 were considered "essential" in a given condition. Genes with dval of >0.01 but 0.1 were considered "compromised" in a given condition, whereas genes with dval >0.1 were considered "fit". A dval ratio was calculated by dividing the dval of a given gene in osteomyelitis by the dval of the same gene during in vitro comparator growth.

RNA isolation and Genechip analysis
For genechip analysis, aerobic cultures of WT or ΔsrrA were prepared as follows. Three colonies of WT or ΔsrrA were inoculated into 10 ml of TSB in a 50 ml Erlenmeyer flask. This culture was grown overnight then back-diluted 1:1000 into 50 ml of TSB in a 250 ml flask. The back-diluted cultures were grown at 37°C and 180 rpm orbital shaking until OD 600 reached 0.5, at which time an equal volume of ice-cold 1:1 acetone:ethanol was added and the cultures were stored at -80°C until processed for RNA isolation. For comparison of RNA from aerobic versus hypoxic conditions, TSB cultures of WT or ΔsrrA were incubated overnight as above, back-diluted 1:1000 into 100 ml of TSB in a 500 ml flask and grown to an OD 600 of 0.5. Fifty milliliters of the culture was then placed into a tightly capped 50ml conical (hypoxic condition) and incubated for one hour at 37°C before mixture with acetone:ethanol and storage at -80°C. The remaining 50 ml of culture was moved to a 250 ml Erlenmeyer flask (aerobic condition) and incubated for one hour at 37°C before mixture with acetone:ethanol and storage at -80°C. For RNA isolation, bacterial cells were harvested by centrifugation and resuspended in LETS buffer (0.1 M LiCl, 10 mM EDTA, 10 mM Tris HCl, 1% SDS). The resuspended cells were disrupted in the presence of 0.5 mm RNAase-free zirconium oxide beads in a Bullet Blender (Next Advance, Averill Park, NY, USA). Disrupted cells were heated at 55°C for 5.5 minutes and centrifuged for 10 minutes at 15,000 rpm. The upper phase was collected and transferred to a new tube before adding 1 ml of TRI-Reagent. After mixing, 200 μl of chloroform was added, and the resultant solution was mixed vigorously for 15 seconds. Samples were centrifuged at 15,000 rpm for 10 min, and the aqueous phase was transferred to a new tube. RNA was precipitated with isopropyl alcohol and subsequently washed with 70% ethanol before drying and resuspension in deionized water. RNA samples were subsequently treated with DNase I and re-purified with a GeneJET RNA Cleanup Kit (Thermo Fisher Scientific, Waltham, MA, USA).
For Genechip analysis, RNA samples were labeled, hybridized to commercially available S. aureus Affymetrix Genechips, and processed as per the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA). Briefly, 10 μg of each RNA sample was reverse transcribed, resulting cDNA was purified using QIAquick PCR Purification Kits (Qiagen, Germantown, MD, USA), fragmented with DNase I (Ambion, Carlsbad, CA, USA), and 3' biotinylated using Enzo Bioarray Terminal Labeling Kits (Enzo Life Sciences, Farmingdale, NY, USA). A total of 1.5 μg of a labeled cDNA sample was hybridized to a S. aureus GeneChip for 16 hr at 45°C, processed, and scanned in an Affymetrix GeneChip 3000 7G scanner as previously described [53,54]. Signal intensity values for each GeneChip qualifier were normalized to the average signal of the microarray to reduce sample labeling and technical variability and the signal for the biological replicates were averaged using GeneSpring GX software (Agilent Technologies, Redwood City, CA, USA) [54][55][56][57]. Differentially expressed transcripts were identified as RNA species that generated a two-fold increase or decrease in WT cells in comparison to ΔsrrA cells during aerobic and hypoxic conditions (t-test, p = 0.05). All related GeneChip data files were deposited in the NCBI Gene Expression Omnibus repository in the MIAME-compliant format.

Supernatant preparations
S. aureus strains were used to inoculate RPMI + 1% CA in glass Erlenmeyer flasks. For aerobic growth, the flask opening was covered lightly with aluminum foil. For hypoxic growth, the flask opening was sealed with a rubber stopper. Cultures were grown for 15 hours. Supernatants were collected after culture centrifugation, and were subsequently filtered through a 0.22 μm filter and concentrated with an Amicon Ultra 3 kDa nominal molecular weight limit centrifugal filter unit (Millipore, Billerica, MA, USA) per the manufacturer's instructions. Following concentration, supernatants were filter sterilized again and frozen at -80°C until used.

Mammalian cell culture and cytotoxicity assays
Primary human osteoblasts were obtained from Lonza (Basel, Switzerland) and cultured per manufacturer's recommendations. All cell lines were obtained from the American Type Culture Collection (ATCC) and propagated at 37°C and 5% CO 2 according to ATCC recommendations. Media was replaced every 2-3 days. All cell culture media was prepared with 1X penicillin/streptomycin and filter sterilized using a 0.22 μm filter prior to use. MC3T3 E-1 cells were cultured in α-MEM, supplemented with 10% fetal bovine serum (FBS). The RAW264.7, Saos-2, and A549 cell lines were grown in Dulbecco's MEM (DMEM) with 10% FBS, McCoy's 5A medium with 15% FBS, and F-12K medium with 10% FBS, respectively. The Jurkat, U937, and HL-60 cell lines were propagated using RPMI with 10% FBS. Cytotoxicity assays were performed in 96-well tissue culture grade plates. Cells were seeded one day prior to intoxication with S. aureus concentrated supernatants or sterile RPMI diluted in the recommended cell culture medium. The following cell densities were used for cytotoxicity assays: MC3T3 E1 murine pre-osteoblastic cells at 5,000 cells per well, primary human osteoblasts at 3,500 cells per well, Saos-2 human osteoblastic cells at 10,000 cells per well, RAW264.7 murine macrophage cells at 10,000 cells per well, A549 lung epithelial cells at 5,000 cells per well, U937 monocytic cells at 15,000 cells per well, HL-60 premyelocytes at 20,000 cells per well, and Jurkat T cells at 50,000 cells per well. Concentrated supernatants were added as dilutions, by mixing between 0.1 μl to 60 μl in a total volume of 200 μl per well to give a dilution spectrum of 0.05%-30% concentrated supernatant (volume/volume). Cell lines in suspension were centrifuged at 3000 x g for 5 minutes prior to intoxication. Cell viability was assessed with CellTiter AQueous One (Promega, Madison, WI, USA) per the manufacturer's instructions at 24 hours post-intoxication.

YFP fluorescence measurements
For fluorescence analysis, overnight cultures of WT and ΔsrrA containing the pDB59 reporter plasmid were back-diluted 1:1000 into 10 ml of RPMI + 1% CA with chloramphenicol in 50ml Erlenmeyer flasks and grown either aerobically or hypoxically as above. YFP was measured using an excitation of 485/20 and emission of 528/20 in a BioTek Synergy HT 96-well plate reader at 0, 6, 9, 12, and 15 hours after back-dilution.

Quantitative RT-PCR
Bacteria were grown for 15 hours as for YFP fluorescence measurements, mixed with 1:1 acetone:ethanol, and stored at -80°C until processed for RNA isolation. RNA isolation was performed as for Genechip analysis. Reverse transcription using 2 μg of RNA and M-MLV reverse transcriptase (Promega, Madison, WI, USA) was performed following the manufacturer's instructions. Quantitative RT-PCR (qRT-PCR) was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, Ca, USA) and the cDNA generated above for each primer pair, including a no reverse transcriptase negative control for 16S rRNA. PCR was conducted on a CFX96 qPCR cycler (Bio-Rad, Hercules, Ca, USA). The cycling program was carried out as recommended by the manufacturer with an annealing temperature of 56°C. Fold-changes were calculated from Ct values averaged from three technical replicates for at least three biological replicates after normalizing to 16S rRNA. The qRT-PCR primer sequences for agrA, hla, and RNAIII were previously published [58]. The qRT-PCR primer sequence for 16S rRNA was also previously published [36].
Supporting Information S1 Table. Genes identified as essential for osteomyelitis by TnSeq analysis. (PDF) S2 Table. Transposon mutants with compromised fitness during osteomyelitis, but not in vitro growth, as identified by TnSeq analysis. (PDF) S3