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

S. aureus Evades Macrophage Killing through NLRP3-Dependent Effects on Mitochondrial Trafficking

Summary: Clinical severity of Staphylococcus aureus respiratory infection correlates with alpha toxin (AT) expression. AT activates the NLRP3 inflammasome; deletion of Nlrp3, or AT neutralization, protects mice from lethal S. aureus pneumonia. We tested the hypothesis that this protection is not due to a reduction in inflammasome-dependent cytokines (IL-1β/IL-18) but increased bactericidal function of macrophages. In vivo, neutralization of AT or NLRP3 improved bacterial clearance and survival, while blocking IL-1β/IL-18 did not. Primary human monocytes were used in vitro to determine the mechanism through which NLRP3 alters bacterial killing. In cells treated with small interfering RNA (siRNA) targeting NLRP3 or infected with AT-null S. aureus, mitochondria co-localize with bacterial-containing phagosomes. Mitochondrial engagement activates caspase-1, a process dependent on complex II of the electron transport chain, near the phagosome, promoting its acidification. These data demonstrate a mechanism utilized by S. aureus to sequester itself from antimicrobial processes within the cell. : In the lung, alpha toxin (AT) is a primary virulence factor used by S. aureus to evade innate immune responses. Cohen et al. demonstrate that AT activation of the NLRP3 inflammasome uncouples key components of the phagocytic killing machinery, namely, mitochondria dissociate from internalized bacteria. Without close association of mitochondria with internalized bacteria, macrophages are unable to effectively kill S. aureus. Keywords: S. aureus, pneumonia, NLRP3 inflammasome, mitochondria, bacterial infection, alpha toxin, monoclonal antibod

Mothers with long QT syndrome are at increased risk for fetal death: findings from a multicenter international study

Background: Most fetal deaths are unexplained. Long QT syndrome is a genetic disorder of cardiac ion channels. Affected individuals, including fetuses, are predisposed to sudden death. We sought to determine the risk of fetal death in familial long QT syndrome, in which the mother or father carries the long QT syndrome genotype. In addition, we assessed whether risk differed if the long QT syndrome genotype was inherited from the mother or father. Objective: This was a retrospective review of pregnancies in families with the 3 most common heterozygous pathogenic long QT syndrome genotypes in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3), which occur in approximately 1 in 2000 individuals. The purpose of our study was to compare pregnancy and birth outcomes in familial long QT syndrome with the normal population and between maternal and paternal carriers of the long QT syndrome genotype. We hypothesized that fetal death before (miscarriage) and after (stillbirths) 20 weeks gestation would be increased in familial long QT syndrome compared with the normal population and that the parent of origin would not affect birth outcomes. Study Design: Our study was a multicenter observational case series of 148 pregnancies from 103 families (80 mothers, 23 fathers) with familial long QT syndrome (60 with LQT1, 29 with LQT2, 14 with LQT3) who were recruited from 11 international centers with expertise in hereditary heart rhythm diseases, pediatric and/or adult electrophysiology, and high-risk pregnancies. Clinical databases from these sites were reviewed for long QT syndrome that occurred in men or women of childbearing age (18–40 years). Pregnancy outcomes (livebirth, stillbirth, and miscarriage), birthweights, and gestational age at delivery were compared among long QT syndrome genotypes and between maternal vs paternal long QT syndrome–affected status with the use of logistic regression analysis. Results: Most offspring (80%; 118/148) were liveborn at term; 66% of offspring (73/110) had long QT syndrome. Newborn infants of mothers with long QT syndrome were delivered earlier and, when the data were controlled for gestational age, weighed less than newborn infants of long QT syndrome fathers. Fetal arrhythmias were observed rarely, but stillbirths (fetal death at >20 weeks gestation) were 8 times more frequent in long QT syndrome (4% vs approximately 0.5%); miscarriages (fetal death at ≤20 weeks gestation) were 2 times that of the general population (16% vs 8%). The likelihood of fetal death was significantly greater with maternal vs paternal long QT syndrome (24.4% vs 3.4%; P=.036). Only 10% of all fetal deaths underwent postmortem long QT syndrome testing; 2 of 3 cases were positive for the family long QT syndrome genotype. Conclusion: This is the first report to demonstrate that mothers with long QT syndrome are at increased risk of fetal death and to uncover a previously unreported cause of stillbirth. Our results suggest that maternal effects of long QT syndrome channelopathy may cause placental or myometrial dysfunction that confers increased susceptibility to fetal death and growth restriction in newborn survivors, regardless of long QT syndrome status

<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

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

<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

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