Juliane Bubeck Wardenburg

Phosphorylation of SLP-76 by the ZAP-70 Protein-tyrosine Kinase Is Required for T-cell Receptor Function

Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-γ1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

Vaccine protection against Staphylococcus aureus pneumonia.

Staphylococcus aureus pneumonia causes significant mortality in hospitalized or healthy individuals, and recent increases in morbidity are attributed to the rapid spread of methicillin-resistant S. aureus (MRSA) strains, which are often not susceptible to antibiotic therapy. α-Hemolysin (Hla), a secreted pore-forming toxin, is an essential virulence factor of MRSA in a mouse model of S. aureus pneumonia. We show that the level of Hla expression by independent S. aureus strains directly correlates with their virulence. Active immunization with a mutant form of Hla (HlaH35L), which cannot form pores, generates antigen-specific immunoglobulin G responses and affords protection against staphylococcal pneumonia. Moreover, transfer of Hla-specific antibodies protects naive animals against S. aureus challenge and prevents the injury of human lung epithelial cells during infection. Thus, Hla vaccination or immunotherapy may prevent S. aureus pneumonia in humans.

Regulation of PAK Activation and the T Cell Cytoskeleton by the Linker Protein SLP-76

Tyrosine phosphorylation of linker proteins enables the T cell antigen receptor (TCR)-associated protein tyrosine kinases to phosphorylate and regulate effector molecules that generate second messengers. We demonstrate here that the SLP-76 linker protein interacts with both nck, an adaptor protein, and Vav, a guanine nucleotide exchange factor for Rho-family GTPases. The assembly of this tri-molecular complex permits the activated Rho-family GTPases to regulate target effectors that interact through nck. In turn, assembly of this complex mediates the enzymatic activation of the p21-activated protein kinase 1 and facilitates actin polymerization. Hence, phosphorylation of linker proteins not only bridges the TCR-associated PTK, ZAP-70, with downstream effector proteins, but also provides a scaffold to integrate distinct signaling complexes to regulate T cell function.

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus α-hemolysin–mediated cellular injury

Staphylococcus aureus α-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, β-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla–ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions.

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.

Bacteria activate sensory neurons that modulate pain and inflammation

Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host–pathogen interactions.

Surface Proteins and Exotoxins Are Required for the Pathogenesis of Staphylococcus aureus Pneumonia

ABSTRACT A model of Staphylococcus aureus-induced pneumonia in adult, immunocompetent C57BL/6J mice is described. This model closely mimics the clinical and pathological features of pneumonia in human patients. Using this system, we defined a role for S. aureus strain Newman surface proteins and secreted exotoxins in pneumonia-related mortality.

Targeting of Alpha-Hemolysin by Active or Passive Immunization Decreases Severity of USA300 Skin Infection in a Mouse Model

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. We tested the hypothesis that alpha-hemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Deltahla) strains caused skin lesions in a mouse infection model that were significantly smaller than those caused by wild-type USA300 and Newman strains. Moreover, infection due to wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Deltahla strains. Passive immunization with Hla-specific antisera or active immunization with a nontoxigenic form of Hla significantly reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude that Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections.

Panton-Valentine Leukocidin Is Not a Virulence Determinant in Murine Models of Community-Associated Methicillin-Resistant Staphylococcus aureus Disease

Increases in the incidence and severity of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have spawned efforts to define unique virulence properties among prevalent strains. Panton-Valentine leukocidin (PVL), a pore-forming cytotoxin, has garnered attention because of its epidemiologic association with CA-MRSA. Using both the clinical isolate LAC, which is representative of the epidemic USA300 strain, and its isogenic PVL-negative strain in murine models of staphylococcal skin infection and pneumonia, we expanded upon recent studies by assessing the contribution of PVL in the genetic background of BALB/c mice. The data presented in this report support the observation that PVL does not contribute to the pathogenesis of staphylococcal infection of mice.