Eric L. Brown

Staphylococcus aureus Panton-Valentine Leukocidin Causes Necrotizing Pneumonia

The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell wall–anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).

Decorin-binding adhesins from Borrelia burgdorferi

Lyme disease is a tick‐transmitted infection caused by the spirochete Borrelia burgdorferi. Ticks deposit B. burgdorferi into the dermis of the host, where they eventually become associated with collagen fibres. We demonstrated previously that B. burgdorferi is unable to bind collagen, but can bind the collagen‐associated proteoglycan decorin and expresses decorin‐binding proteins (Dbps). We have now cloned and sequenced two genes encoding the proteins, DbpA and DbpB, which have a similar structure, as revealed by circular dichroism (CD) spectroscopy of recombinant proteins. Competition experiments revealed a difference in binding specificity between DbpA and DbpB. Western blot analysis of proteinase K‐treated intact B. burgdorferi and transmission electron microscopy studies using antibodies raised against recombinant Dbps demonstrated that these proteins are surface exposed. DbpA effectively inhibits the attachment of B. burgdorferi to a decorin substrate, whereas DbpB had a marginal effect, suggesting a difference in substrate specificity between the two Dbps. Polystyrene beads coated with DbpA adhered to a decorin‐containing extracellular matrix produced by cultured skin fibroblasts, whereas beads coated with OspC did not. Taken together, these data suggest that Dbps are adhesins of the MSCRAMM (microbial surface component‐recognizing adhesive matrix molecule) family, which mediate B. burgdorferi attachment to the extracellular matrix of the host.

The Panton-Valentine leukocidin vaccine protects mice against lung and skin infections caused by Staphylococcus aureus USA300.

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton-Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl-deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.

Role of Complement Activation in a Model of Adult Respiratory Distress Syndrome

The adult respiratory distress syndrome is characterized by arterial hypoxemia as a result of increased alveolar capillary permeability to serum proteins in the setting of normal capillary hydrostatic pressures. Because bacterial sepsis is prominent among the various diverse conditions associated with altered alveolar capillary permeability, we studied the effect of bacteremia with attendant complement activation on the sequestration of microorganisms and the leakage of albumin in the lungs of guinea pigs. Pneumococci were injected intravenously into guinea pigs and their localization was studied. Unlike normal guinea pigs, complement-depleted guinea pigs did not localize injected bacteria to the lungs. Preopsonization of organisms did not correct this defect in pulmonary localization of bacteria in complement-depleted animals, suggesting that a fluid-phase component of complement activation was required. Genetically C5-deficient mice showed no pulmonary localization of bacteria. C5-sufficient mice demonstrated the usual pulmonary localization, thus further suggesting that the activation of C5 might be important in this localization. The infusion of activated C5 increased alveolar capillary permeability to serum proteins as assayed by the amount of radioactive albumin sequestered in the lung. Neutropenic animals did not develop altered capillary permeability after challenge with activated C5. Thus, complement activation through C5, in the presence of neutrophils, induces alterations in pulmonary alveolar capillary permeability and causes localization of bacteria to the pulmonary parenchyma. Complement activation in other disease states could potentially result in similar clinical manifestations.

Identification and Characterization of the C3 Binding Domain of the Staphylococcus aureus Extracellular Fibrinogen-binding Protein (Efb)*

The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.24 μm for the Efb-C3d binding using intrinsic fluorescence quenching assays. Using truncated, recombinant forms of Efb, we also demonstrate that the C3b binding region of Efb is located within the C terminus, in contrast to the fibrinogen binding domains that are located at the N-terminal end of the protein. Enzyme-linked immunosorbent assay-type binding assays demonstrated that recombinant Efb could bind to both C3b and fibrinogen simultaneously, forming a trimolecular complex and that the C-terminal region of Efb could inhibit complement activity in vitro. In addition, secondary structure analysis using circular dichroism spectroscopy revealed that the C-terminal, C3b binding region of Efb is composed primarily of α-helices, suggesting that this domain of Efb represents a novel type of C3b-binding protein.

Inhibition of Complement Activation by a Secreted Staphylococcus aureus Protein

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.

A role for decorin in cutaneous wound healing and angiogenesis

Decorin is known to influence tissue tensile strength and cellular phenotype. Therefore, decorin is likely to have an impact on tissue repair, including cutaneous wound healing. In this study, cutaneous healing of both excisional and incisional full‐thickness dermal wounds was studied in decorin‐deficient (Dcn−/−) animals. A statistically significant delay in excisional wound healing in the Dcn−/− mice occurred at 4 and 10 days postwounding and, in incisional wounds at 4, 10, and 18 days when compared with wild‐type (Dcn−/−) controls. Fibrovascular invasion into polyvinylalcohol sponges was significantly increased by day 18 in Dcn−/− mice relative to Dcn+/+ mice. The 18‐day sponge implants in the Dcn−/− mice showed a marked accumulation of biglycan when compared with the corresponding implants in Dcn+/+ mice. Thus, regulated production of decorin may serve as an excellent therapeutic approach for modifying impaired wound healing and harmful foreign body reactions.

Resistance to Lyme disease in decorin-deficient mice

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Protective niche for Borrelia burgdorferi to evade humoral immunity.

The Lyme disease spirochete, Borrelia burgdorferi, is an extracellular microbe that causes persistent infection despite the development of strong immune responses against the bacterium. B. burgdorferi expresses several ligand-binding lipoproteins, including the decorin-binding proteins (Dbps) A and B, which may mediate attachment to decorin, a major component of the host extracellular matrix during murine infection. We show that B. burgdorferi was better protected in the joints and skin, two tissues with a higher decorin expression, than in the urinary bladder and heart, two tissues with a lower decorin expression, during chronic infection of wild-type mice. Targeted disruption of decorin alone completely abolished the protective niche in chronically infected decorin-deficient mice but did not affect the spirochete burden during early infection. The nature of protection appeared to be specific because the spirochetes with higher outer surface protein C expression were not protected while the protective niche seemed to favor the spirochetes with a higher dbpA expression during chronic infection. These data suggest that spirochetal DbpA may interact with host decorin during infection and such interactions could be a mechanism that B. burgdorferi uses to evade humoral immunity and establish chronic infection.

Virulence potential of the staphylococcal adhesin CNA in experimental arthritis is determined by its affinity for collagen.

BACKGROUND Staphylococcus aureus is a major cause of bacterial arthritis, which often results in severe joint damage. CNA, a collagen adhesin of S. aureus, was shown to be a virulence factor in several animal models. However, the precise molecular mechanism by which CNA contributes to virulence remains unclear. METHODS We examined the role of the collagen-binding function of CNA in a mouse model of septic arthritis by comparing the virulence of isogenic strains of S. aureus expressing (1) wild-type CNA, (2) a truncated form of CNA (CNA35) with a higher affinity for collagen than the wild type, (3) CNA35 containing a single point mutation resulting in loss of collagen binding, (4) CNA lacking the collagen-binding domain, and (5) the collagen-binding domain of ACE (adhesin of collagen from Enterococcus faecalis). RESULTS AND CONCLUSIONS The results provide, for the first time, direct evidence that the virulence of CNA depends on its collagen-binding ability. Collagen binding facilitated early colonization of the joints of mice. Furthermore, the virulence potential of the adhesin is determined by the adhesins affinity for its ligand, as well as its binding kinetics.