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.

Targeting agr- and agr-Like quorum sensing systems for development of common therapeutics to treat multiple gram-positive bacterial infections.

Invasive infection by the Gram-positive pathogen Staphylococcus aureus is controlled by a four gene operon, agr that encodes a quorum sensing system for the regulation of virulence. While agr has been well studied in S. aureus, the contribution of agr homologues and analogues in other Gram-positive pathogens is just beginning to be understood.

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.

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.

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.

Confinement-induced quorum sensing of individual Staphylococcus aureus bacteria.

It is postulated that in addition to cell density, other factors such as the dimensions and diffusional characteristics of the environment could influence quorum sensing (QS) and induction of genetic reprogramming. Modeling studies predict that QS may operate at the level of a single cell, but, owing to experimental challenges, the potential benefits of QS by individual cells remain virtually unexplored. Here we report a physical system that mimics isolation of a bacterium, such as within an endosome or phagosome during infection, and maintains cell viability under conditions of complete chemical and physical isolation.

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.

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.

Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen. In response to changing host environments, this bacterium has the capability to switch on selective sets of genes to enhance its chances for survival. This switching process is precisely controlled by global regulatory elements.

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.

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.

The Genomic Aspect of Virulence, Sepsis, and Resistance to Killing Mechanisms in Staphylococcus aureus.

Staphylococcus aureus is widely appreciated as a pathogen, despite the fact that this microorganism is usually a benign colonizer of the host, rarely if ever causing infection. However, this bacterium, in response to changing environments, will occasionally switch from a commensal to a lethal pathogen. S.

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.

Inhibition of beta 2 integrin receptor and Syk kinase signaling in monocytes by the Src family kinase Fgr.

While beta 2 integrin ligand-receptor recognition interactions are well characterized, less is known about how these events trigger signal transduction cascades to regulate the transition from tethering to firm adhesion, spreading, and transendothelial migration. We have identified critical positive and negative regulatory components of this cascade in monocytes. Whereas the Syk tyrosine kinase is essential for beta 2 integrin signaling and cell spreading, the Src family kinase Fgr is a negative regulator of this pathway.

An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(o)-mediated signal transduction.

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE.

CD47-signal regulatory protein alpha (SIRPalpha) regulates Fcgamma and complement receptor-mediated phagocytosis.

In autoimmune hemolytic anemia (AIHA), circulating red blood cells (RBCs) opsonized with autoantibody are recognized by macrophage Fcgamma and complement receptors. This triggers phagocytosis and elimination of RBCs from the circulation by splenic macrophages. We recently found that CD47 on unopsonized RBCs binds macrophage signal regulatory protein alpha (SIRPalpha), generating a negative signal that prevents phagocytosis of the unopsonized RBCs.

Serum amyloid P component and C-reactive protein mediate phagocytosis through murine Fc gamma Rs.

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are acute-phase serum proteins in mice and humans, respectively. Although SAP binds to DNA and chromatin and affects clearance of these autoantigens, no specific receptor for SAP has been identified. CRP is an opsonin, and we have shown that it binds to FcgammaR.

Role of CD47 as a marker of self on red blood cells.

The immune system recognizes invaders as foreign because they express determinants that are absent on host cells or because they lack "markers of self" that are normally present. Here we show that CD47 (integrin-associated protein) functions as a marker of self on murine red blood cells. Red blood cells that lacked CD47 were rapidly cleared from the bloodstream by splenic red pulp macrophages.

Survival of Staphylococcus aureus inside neutrophils contributes to infection.

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S.

Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr.

Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages.

Intracellular demonstration of active TGFbeta1 in B cells and plasma cells of autoimmune mice. IgG-bound TGFbeta1 suppresses neutrophil function and host defense against Staphylococcus aureus infection.

Infection remains a leading cause of morbidity and mortality in patients with SLE. To investigate this, previously we assessed the host defense status of autoimmune MRL/lpr mice and found that elaboration of active TGFbeta suppressed neutrophil function and decreased survival in response to Staphylococcus aureus infection. The purpose of the present work was to elucidate the molecular form and the cellular source of the active TGFbeta involved.

Domain-specific interactions between entactin and neutrophil integrins. G2 domain ligation of integrin alpha3beta1 and E domain ligation of the leukocyte response integrin signal for different responses.

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors.

Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice.

Granulocyte [polymorphonuclear leucocyte (PMN)] migration to sites of infection and subsequent activation is essential for host defense. Gene-targeted mice deficient for integrin-associated protein (IAP, also termed CD47) succumbed to Escherichia coli peritonitis at inoccula survived by heterozygous littermates. In vivo, they had an early defect in PMN accumulation at the site of infection.

Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding.

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg-Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn-coated particles to many cells expressing alpha v beta 3.