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.

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.

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.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

Apolipoprotein B Is an innate barrier against invasive Staphylococcus aureus infection.

Staphylococcus aureus is both a colonizer of humans and a cause of severe invasive infections. Although the genetic basis for phenotype switching from colonizing to invasive has received significant study, knowledge of host factors that antagonize the switch is limited. We show that VLDL and LDL lipoproteins interfere with this switch by antagonizing the S. aureus agr quorum-sensing system that upregulates genes required for invasive infection. The mechanism of antagonism entails binding of the major structural protein of these lipoproteins, apolipoprotein B, to an S. aureus autoinducing pheromone, preventing attachment of this pheromone to the bacteria and subsequent signaling through its receptor, AgrC. Mice deficient in plasma apolipoprotein B, either genetically or pharmacologically, are more susceptible to invasive agr+ bacterial infection, but not to infection with an agr deletion mutant. Therefore, apolipoprotein B at homeostatic levels in blood is an essential innate defense effector against invasive S. aureus infection.

Inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: new role for the NADPH oxidase in host defense.

Quorum sensing triggers virulence factor expression in medically important bacterial pathogens in response to a density-dependent increase in one or more autoinducing pheromones. Here, we show that phagocyte-derived oxidants target these autoinducers for inactivation as an innate defense mechanism of the host. In a skin infection model, expression of phagocyte NADPH oxidase, myeloperoxidase, or inducible nitric oxide synthase was critical for defense against a quorum-sensing pathogen, Staphylococcus aureus, but not for defense against a quorum sensing-deficient mutant. A virulence-inducing peptide of S. aureus was inactivated in vitro and in vivo by reactive oxygen and nitrogen intermediates, including HOCl and ONOO(-). Inactivation of the autoinducer prevented both the up-regulation of virulence gene expression and the downstream sequelae. MS analysis of the inactivated peptide demonstrated that oxidation of the C-terminal methionine was primarily responsible for loss of activity. Treatment of WT but not NADPH oxidase-deficient mice with N-acetyl methionine to scavenge the inhibitory oxidants increased in vivo quorum sensing independently of the bacterial burden at the site of infection. Thus, oxidant-mediated inactivation of an autoinducing peptide from S. aureus is a critical innate defense mechanism against infection with this pathogen.

Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation.

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including alpha hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.

Role for plastin in host defense distinguishes integrin signaling from cell adhesion and spreading.

Integrin ligation activates both cell adhesion and signal transduction, in part through reorganization of the actin cytoskeleton. Plastins (also known as fimbrins) are actin-crosslinking proteins of the cortical cytoskeleton present in all cells and conserved from yeast to mammals. Here we show that plastin-deficient polymorphonuclear neutrophils (PMN) are deficient in killing the bacterial pathogen Staphylococcus aureus in vivo and in vitro, despite normal phagocytosis. Like integrin beta2-deficient PMN, plastin-deficient PMN cannot generate an adhesion-dependent respiratory burst, because of markedly diminished integrin-dependent syk activation. Unlike beta2(-/-) PMN, plastin-deficient PMN adhere and spread normally. Deficiency of plastin thus separates the classical integrin receptor functions of adhesion and spreading from intracellular signal transduction.

Lethal autoimmune hemolytic anemia in CD47-deficient nonobese diabetic (NOD) mice.

The glycoprotein CD47 (integrin-associated protein, IAP) is present on the surface of virtually all cells, including red blood cells (RBCs). CD47 acts like a marker of self by ligating the macrophage inhibitory receptor signal regulatory protein alpha (SIRPalpha). In this manner mild reactivity of wild-type RBCs with macrophage phagocytic receptors is tolerated, whereas otherwise identical CD47-deficient RBCs are rapidly eliminated. We show here that virtually all CD47-deficient nonobese diabetic (NOD) mice spontaneously develop severe lethal autoimmune hemolytic anemia (AIHA) at 180 to 280 days of age, whereas none of the control CD47(+) NOD mice develop lethal AIHA at least during the first year of life. This phenotype is at least partially due to a markedly increased rate of elimination of opsonized CD47(-/-) compared to CD47(+) RBCs. Similarly, CD47(-/-)C57BL/6 mice were much more sensitive than their wild-type counterparts to experimental passive AIHA induced by anti-RBC monoclonal antibodies. Thus, CD47-SIRPalpha signaling can have a profound influence on the severity of AIHA, making manipulation of this signaling pathway a theoretically appealing avenue in the treatment of the disease.