Jon K. Femling

Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance.

Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.

The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2^{.}}^{-}\end{equation*}\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2^{.}}^{-}\end{equation*}\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.

Neutrophil Bleaching of GFP-Expressing Staphylococci: Probing the Intraphagosomal Fate of Individual Bacteria

Successful host defense against bacteria such as Staphylococcus aureus (SA) depends on a prompt response by circulating polymorphonuclear leukocytes (PMN). Stimulated PMN create in their phagosomes an environment inhospitable to most ingested bacteria. Granules that fuse with the phagosome deliver an array of catalytic and noncatalytic antimicrobial peptides, while activation of the NADPH oxidase at the phagosomal membrane generates reactive oxygen species within the phagosome, including hypochlorous acid (HOCl), formed by the oxidation of chloride by the granule protein myeloperoxidase in the presence of H2O2. In this study, we used SA-expressing cytosolic GFP to provide a novel probe of the fate of SA in human PMN. PMN bleaching of GFP in SA required phagocytosis, active myeloperoxidase, H2O2 from the NADPH oxidase, and chloride. Not all ingested SA were bleached, and the number of cocci within PMN-retaining fluorescent GFP closely correlated with the number of viable bacteria remaining intracellularly. The percent of intracellular fluorescent and viable SA increased at higher multiplicity of infection and when SA presented to PMN had been harvested from the stationary phase of growth. These studies demonstrate that the loss of GFP fluorescence in ingested SA provides a sensitive experimental probe for monitoring biochemical events within individual phagosomes and for identifying subpopulations of SA that resist intracellular PMN cytotoxicity. Defining the molecular basis of SA survival within PMN should provide important insights into bacterial and host properties that limit PMN antistaphylococcal action and thus contribute to the pathogenesis of staphylococcal infection.

Synergy between extracellular group IIA phospholipase A2 and phagocyte NADPH oxidase in digestion of phospholipids of Staphylococcus aureus ingested by human neutrophils.

Acute inflammatory responses to invading bacteria such as Staphylococcus aureus include mobilization of polymorphonuclear leukocytes (PMN) and extracellular group IIA phospholipase A2 (gIIA-PLA2). Although accumulating coincidentally, the in vitro anti-staphylococcal activities of PMN and gIIA-PLA2 have thus far been studied separately. We now show that degradation of S. aureus phospholipids during and after phagocytosis by human PMN requires the presence of extracellular gIIA-PLA2. The concentration of extracellular gIIA-PLA2 required to produce bacterial digestion was reduced 10-fold by PMN. The effects of added gIIA-PLA2 were greater when present before phagocytosis but even apparent when added after S. aureus were ingested by PMN. Related group V and X PLA2, which are present within PMN granules, do not contribute to bacterial phospholipid degradation during and after phagocytosis even when added at concentrations 30-fold higher than that needed for action of the gIIA-PLA2. The action of added gIIA-PLA2 required catalytically active gIIA-PLA2 and, in PMN, a functional NADPH oxidase but not myeloperoxidase. These findings reveal a novel collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

ω-Hydroxyemodin Limits Staphylococcus aureus Quorum Sensing-Mediated Pathogenesis and Inflammation

ABSTRACT Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation.

Nox2 Modification of LDL Is Essential for Optimal Apolipoprotein B-mediated Control of agr Type III Staphylococcus aureus Quorum-sensing

Staphylococcus aureus contains an autoinducing quorum-sensing system encoded within the agr operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within agr has generated four agr specific groups, agr I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives agr signaling. Because agr signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize agr signaling. We determined whether two host defense factors that inhibit AIP1-induced agrI signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced agrIII signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against agrIII signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against S. aureus agrIII infection by binding the secreted signaling peptide, AIP3, and preventing expression of the agr-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes agr signaling by all four agr alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against agr signaling. These data reveal a novel role for oxLDL in host defense against S. aureus quorum-sensing signaling.

EMS patients and walk-in patients presenting with severe sepsis: differences in management and outcome.

Objectives Sepsis is a significant problem. The differences between patients with sepsis who walk into the emergency department (ED) and those who are transported via emergency medical services (EMS) have not been clarified. The aim of the study was to determine whether there was a difference in outcome between patients arriving by EMS and those presenting directly to the ED. Methods We prospectively collected and reviewed a cohort of all cases of severe sepsis and septic shock admitted to the medical intensive care unit from the ED from November 2009 to March 2012. Extracted data were basic demographic information (including mode of ED arrival), clinical data, and treatments. We calculated Systemic Inflammatory Response Syndrome criteria, Acute Physiology and Chronic Health Evaluation II scores, and Sequential Organ Failure Assessment (SOFA) scores. The primary outcome was mortality in severely ill patients with sepsis. Results A total of 378 subjects (78%) presented by EMS and 107 subjects were walk-in patients (22%). Patients transported via EMS were older (P < 0.01), had fewer lactates >4 (P < 0.02), a more altered mental status (P < 0.01), and higher respiratory rates (P < 0.05) than did walk-in patients. Patients transported by EMS had worse disease severity when measured by an Acute Physiology and Chronic Health Evaluation II score (P < 0.01) but not by SOFA score. EMS patients had a shorter time to receiving antibiotics (P = 0.02) and central line placement (P < 0.01) than did walk-in patients. In a logistic model, mortality was associated with increasing age (adjusted odds ratio 1.3; 95% confidence interval [CI] 1.2–1.4), higher first-measured ED lactates (1.2; 95% CI 1.1–1.2), and increased initial SOFA score (adjusted odds ratio 1.2; 95% CI 1.1–1.3) but not EMS arrival or prehospital fluids. Conclusions Neither arrival by EMS nor fluid administration by EMS is associated with decreased mortality in severe sepsis.

Nosocomial Infections after Severe Trauma Are Associated With Lower Apolipoproteins B and AII

BACKGROUND Infection after severe trauma is a significant cause of morbidity and mortality days to weeks after the initial injury. Apolipoproteins play important roles in host defense and circulating concentrations are altered by the acute inflammatory response. The purpose of this study was to determine if patients who acquire infection after severe trauma have significantly lower apolipoprotein levels than trauma patients who do not become infected. METHODS We conducted a case–control study on a prospectively identified cohort of adult patients admitted to our intensive care unit after severe trauma (Injury Severity Score ≥ 16). We compared plasma apolipoprotein levels between patients who acquired an infection within 30 days after trauma (cases) and those that remained infection free (controls). RESULTS Of 40 patients experiencing severe trauma, we identified 22 cases that developed an infection within 30 days after injury. Cases had significantly lower posttrauma plasma levels of apolipoprotein B (p = 0.02) and apolipoprotein AII (p = 0.02) compared with controls. Consistent with previous studies, cases also received greater volumes of crystalloid infusions (p < 0.01) and blood transfusions (p < 0.01). Cases also had a more profound inflammatory response as measured by interleukin 6 levels (p = 0.02). CONCLUSION Infection after severe trauma is associated with decreased circulating apolipoproteins as compared with uninfected controls. Profoundly decreased plasma apolipoproteins B and AII could potentially contribute to the impaired immunity after severe trauma. Apolipoproteins are potential targets for identifying those patients at risk of infection after trauma and for interventions aimed at preventing nosocomial infections. LEVEL OF EVIDENCE Prognostic study, level III.

Rim-to-Rim Wearables at the Canyon for Health (R2R WATCH): Experimental Design and Methodology

The Rim-to-Rim Wearables At The Canyon for Health (R2R WATCH) study examines metrics recordable on commercial off the shelf (COTS) devices that are most relevant and reliable for the earliest possible indication of a health or performance decline. This is accomplished through collaboration between Sandia National Laboratories (SNL) and The University of New Mexico (UNM) where the two organizations team up to collect physiological, cognitive, and biological markers from volunteer hikers who attempt the Rim-to-Rim (R2R) hike at the Grand Canyon. Three forms of data are collected as hikers travel from rim to rim: physiological data through wearable devices, cognitive data through a cognitive task taken every 3 hours, and blood samples obtained before and after completing the hike. Data is collected from both civilian and warfighter hikers. Once the data is obtained, it is analyzed to understand the effectiveness of each COTS device and the validity of the data collected. We also aim to identify which physiological and cognitive phenomena collected by wearable devices are the most relatable to overall health and task performance in extreme environments, and of these ascertain which markers provide the earliest yet reliable indication of health decline. Finally, we analyze the data for significant differences between civilians’ and warfighters’ markers and the relationship to performance. This is a study funded by the Defense Threat Reduction Agency (DTRA, Project CB10359) and the University of New Mexico (The main portion of the R2R WATCH study is funded by DTRA. UNM is currently funding all activities related to bloodwork. DTRA, Project CB10359; SAND2017-1872 C). This paper describes the experimental design and methodology for the first year of the R2R WATCH project.

Innate Sex Bias of Staphylococcus aureus Skin Infection Is Driven by α-Hemolysin

Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to S. aureus SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for S. aureus SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of S. aureus dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the S. aureus–secreted virulence factor α-hemolysin (Hla). Infection with wild-type S. aureus suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic hla deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing S. aureus, demonstrated sex-specific S. aureus bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting S. aureus SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against S. aureus skin infection.