<i>S</i>. <i>aureus</i> modulates quorum sensing and exotoxin production in response to oxygenation.

<p>Supernatants from WT or Δ<i>srrA</i> 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 <i>agr</i>P3-dependent YFP expression in WT or Δ<i>srrA</i> 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 <i>p</i><0.05 and 0.01, respectively relative to WT aerobic at 15 hours as determined by Student’s <i>t</i> test. # represents <i>p</i><0.05 relative to Δ<i>srrA</i> aerobic. (C) cDNA samples from WT or Δ<i>srrA</i> 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 <i>p</i><0.05, ** denotes <i>p</i><0.01, and *** denotes <i>p</i><0.001 relative to WT aerobic.</p

<i>S</i>. <i>aureus</i> osteomyelitis triggers reduced oxygen availability in skeletal tissues.

<p>Oxygen tension (pO₂) was measured in murine femurs infected with <i>S</i>. <i>aureus</i> (black circles) at 1, 4, 7 and 10 days post-infection (n = 6 from two independent experiments). Uninfected femurs (open squares) were measured for oxygen tension immediately following (n = 5) or 4 days after (n = 3) a mock inoculation procedure. Oxygen tension is reported as mmHg. Horizontal lines represent the mean. Error bars represent the SD. Dotted line represents the upper limit of detection.</p

Neutrophil depletion rescues the intraosseous growth defect of an <i>srrA</i> mutant.

<p>Mice were given serial injections of anti-Ly6G monoclonal antibody. As a control, mice received injections of an isotype control antibody. At 14 days post-infection, femurs were processed for CFU enumeration. N = 4 mice per group. Horizontal lines represent the mean. Error bars represent SD. Significance determined by Students <i>t</i> test.</p

SrrAB is required for intraosseous survival and cortical bone destruction during <i>S</i>. <i>aureus</i> osteomyelitis.

<p>Osteomyelitis was induced in groups of mice using WT or Δ<i>srrA</i> strains. (A) At 5 and 14 days post-infection, femurs were processed for colony forming units (CFU) enumeration. N = 5 mice per group. Horizontal line represents the mean and error bars represent SD. (B and C) Antero-posterior views of WT (B) or Δ<i>srrA</i> (C) infected femurs at 14 days post-inoculation. (D) MicroCT imaging analysis of cortical bone destruction (mm³) 14 days post-inoculation. N = 4 mice per group. Error bars represent the SEM. Statistical significance determined by Students <i>t</i> test.</p

The <i>srrAB</i> promoter is active in hypoxic skeletal tissues.

<p>Groups of mice (n = 3 per group) were subjected to osteomyelitis by infection with WT bacteria containing either P<i>srrAB</i>-pAmiLux or pAmiLux (promoterless control). At 1 or 24 hours post-inoculation, infected femurs were explanted and immediately imaged on an IVIS 200 system (5 minute exposure).</p