Carl H. Hammer

Identification of a Novel Staphylococcus aureus Two- Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Community‐associated methicillin‐resistant Staphylococcus aureus (CA‐MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA‐MRSA pathogenesis are poorly understood and a comprehensive analysis of CA‐MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2‐dimensional gel electrophoresis coupled with automated direct infusion‐tandem mass spectrometry (ADI‐MS/MS) analysis. Eleven known virulence‐related exoproteins differed in abundance between the strains, including alpha‐haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1‐phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA‐MRSA as a human pathogen.

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.

In Vitro Proteolytic Processing of the MD145 Norovirus ORF1 Nonstructural Polyprotein Yields Stable Precursors and Products Similar to Those Detected in Calicivirus-Infected Cells

ABSTRACT The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q330/G331, Q696/G697, E875/G876, E1008/A1009, and E1189/G1190) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems.

A quantitative analysis of the interactions of antipneumococcal antibody and complement in experimental pneumococcal bacteremia.

The mechanism of protection of type-specific antipneumococcal antibody and complement in bacteremia was investigated with purified rabbit antibody and a guinea pig model of pneumococcal bacteremia. IgG and IgM were isolated from the sera of rabbits immunized with type 7 pneumococci (Pn), and their binding to Pn was quantitated. The number of antibody-binding sites on the pnuemococcal capsule was also determined. Pn were incubated with various amounts of the immunoglobulin preparations before intravenous injection into nonimmune guinea pigs. Whereas 120 molecules of IgM per Pn were sufficient to enhance bloodstream clearance of Pn, 1,400 molecules of IgG per bacterium were required to produce this effect. As the amount of either IgG or IgM added to the Pn was increased, the rate of bloodstream clearance accelerated. In striking contrast, greater than 1,000 molecules of IgM had no effect on the rate of clearance in C4-deficient guinea pigs, which cannot activate complement via the classic pathway. Similarly, 5,000 molecules of IgG had only minimal effect in C4-deficient guinea pigs, and 24,000 molecules of IgG had no effect in guinea pigs depleted of complement by cobra venom factor. Thus, the in vivo opsonic effects of both IgG and IgM anticapsular antibody are mediated via their ability to activate complement. IgG anti-pneumococcal cell wall antibody, raised by intravenous injection of rabbits with unencapsulated Pn, had no effect on the rate of bloodstream clearance of Pn or on the polymorphonuclear leukocyte killing of type 7 Pn in an in vitro bacterial assay. Because the opsonic effects of anticapsular antibody required complement activation, the ability of anticell wall IgG to activate complement was compared with the two classes of anticapsular antibody. As judged by depletion of C3 and C4 from guinea pig serum, as well as by the fixation of radiolabeled C3 to Pn, IgM anticapsular antibody was the best complement activator. However, anticell wall IgG was somewhat more active than anticapsular IgG in each of these tests of complement activation and fixation. When equivalent amounts of C3 were fixed to Pn by each of the three antibodies, Pn sensitized with IgG and IgM anticapsular antibodies caused immune adherence, whereas Pn sensitized with anticell wall IgG did not. This may explain the failure of anticell wall antibody of mediate complement-dependent phagocytosis of Pn in vivo or in vitro. Although anticell wall IgG is capable of activating complement and fixing C3 to Pn, it is not opsonic; the most likely reason is that the nonopsonic antibody mediates C3 deposition in sites on the Pn that cannot interact efficiently with phagocytic cell C3 receptors.

Studies of human C5a as a mediator of inflammation in normal human skin.

C5a is an 11,000-D fragment of the fifth component of complement (C5) with potent anaphylatoxic and leukocyte chemotactic activities. C5a is believed to play an important role in the pathophysiology of certain skin disorders and systemic diseases with cutaneous manifestations. However, there is very little known about the in vivo reactivity of C5a in man. In this study we examined the effects of intradermal injections of human C5a in 17 normal volunteers. C5a was prepared by interacting highly purified human C5 with zymosan bound alternative pathway C5 convertase under conditions resulting in consumption of approximately 90% of the C5 substrate. C5a produced in this manner was chemotactic for human neutrophils and monocytes (0.5 X 10(-7) to 10(-9) M) and caused neutrophil aggregation and myeloperoxidase release (concentrations greater than or equal to 10(-10) M) in vitro. In vivo, C5a produced immediate wheal and flare reactions in all volunteers, and was active in doses as low as 1 ng (10(-13) mol). Intradermal testing with 20 ng of C5a in eight volunteers produced a maximal mean wheal of 11.75 mm (+/- 0.80 mm SEM) 20 min after anaphylatoxin injection, and a maximal mean erythema of 62.50 mm (+/- 3.27 mm SEM) 10 min after C5a administration. Reactions at C5a test sites were dose-related, associated with marked pruritus in some subjects, resolved without lesion formation, and were not associated with late phase reactions. In vivo testing revealed that human C5a was a more potent mediator of wheal and flare reactions than histamine, 48/80, human C3a, or morphine sulfate. Skin biopsies from eight volunteers 20 min after intradermal injection of 20 ng of C5a revealed a neutrophil-predominant perivascular infiltrate, endothelial cell edema, and sites of leukocytoclasis. Mast cell degranulation was observed on both light and electron microscopy of biopsies from C5a test sites. Although erythema at C5a injection sites was reduced by pretreating volunteers with hydroxyzine, whealing reactions and cellular infiltrates in biopsies were unaffected by this H1-antihistamine. Moderate doses of systemic corticosteroids did not alter clinical or histologic reactions at C5a injection sites in two volunteers. This study, using doses within the potential physiologic range of the anaphylatoxin, provides a comprehensive assessment of the effect of human C5a on normal human skin.

Immunoregulatory disorders associated with hereditary angioedema: I. Clinical manifestations of autoimmune disease

Occasional reports have appeared linking hereditary angioedema (HAE) with autoimmune diseases. We have systematically evaluated 157 patients for manifestations of autoimmunity. Nineteen of these patients (12%) had clinical immunoregulatory diseases including glomerulonephritis (five patients), Sjögrens syndrome (three), inflammatory bowel disease (three), thyroiditis (two), systemic lupus erythematosus (one), drug-induced lupus (one), rheumatoid arthritis (one), juvenile rheumatoid arthritis with IgA deficiency (one), incipient pernicious anemia (one), and sicca syndrome (one). All eight patients with HAE who developed an autoimmune disease with a known human histocompatibility antigen association developed a disease associated with their histocompatibility antigen haplotype (p = 0.014). Although only four patients developed Sjögrens syndrome or sicca syndrome, an additional nine manifested part of the sicca complex. We also found patients with HAE with features suggestive of an immune-based abnormality. These features included idiopathic pancreatitis (three patients), Raynauds disease (two), partial lipodystrophy (one), chronic chorioretinitis (one), and alopecia universalis (one).

A new simplified procedure for C1 inhibitor purification. A novel use for jacalin-agarose.

C1 inhibitor (C1-INH), the major regulatory protein of the classical pathway of complement activation, is also involved in the regulation of several other plasma proteolytic systems including the coagulation, fibrinolytic and contact systems. All the previously published methods for the purification of C1-INH are time-consuming and some do not yield highly pure protein. Recently, it was reported that Jack fruit (Artocarpus integrifolia) lectin, also called jacalin, binds C1-INH. Since jacalin binds only a small number of human serum proteins it appeared that jacalin-agarose affinity chromatography would constitute a very selective early step for the purification of C1-INH. Consequently we have designed a new, simplified three-step procedure for the purification of C1-INH which includes PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose which takes advantage of the marked hydrophilicity of the inhibitor. This procedure has three major advantages over those which have been the most frequently used. Firstly, it includes only two fast chromatographic steps. Secondly, because the C1-INH pool is cleanly and predictably separated from the unwanted proteins by differential elution conditions in both chromatographic steps, no antigenic or functional assays are required to define the desired peaks. Thirdly, only the final product is dialyzed while all other methods required several buffer changes. For these reasons this procedure is much faster and simpler than the previously published methods. About 10-12 mg of highly purified and fully active C1-INH can be obtained within 1 day from 120 ml of plasma giving an average yield of 40-45%. This method may thus be highly adaptable to bulk purification for clinical use or for preparation of genetically or pathologically altered C1-INH from clinical specimens.

Immunoregulatory disorders associated with hereditary angioedema: II. Serologic and cellular abnormalities

Hereditary angioedema is defined biochemically by a deficiency in the functional activity of the inhibitor of Cl, Cl esterase inhibitor (Cl INH). Deficiency of this regulator of the early classic pathway of complement results in chronic activation of this cascade with a resultant deficiency of C4 and C2. Ninety-seven patients with either complicated (associated with autoimmune disorders) or uncomplicated hereditary angioedema were evaluated for laboratory evidence of immunoregulatory defects. Specific cellular and humoral abnormalities were found and included increased mean total lymphocyte counts, increased mean Leu 4+ (total) and Leu 3+ (helper) T cells, an increased mean Leu 3/Leu 2 (helper/suppressor T cell) ratio, polyclonal B cell activation, and evidence of circulating immune complexes. C4 functional titers were negatively correlated with percent Leu 3+ cells and absolute Leu 3+ cell numbers. We failed to detect any evidence of immune deficiency in this population, and yet a statistically significant number of patients demonstrated elevated levels of antibodies to Epstein-Barr virus antigens when patients were compared to a control group. Thus, early classic complement pathway activation and/or partial complement component deficiency may effect T cell subpopulations and B cell activation. However, additional predisposing factors (e.g., genetic or viral) appear necessary for the development of a particular autoimmune disease in hypocomplementemic patients.

Acquired angioedema: Observations on the mechanism of action of autoantibodies directed against C1 esterase inhibitor

We describe a mechanism of action of autoantibodies reactive with the C1 esterase inhibitor (C1EI) molecule found in three patients with acquired angioedema without associated diseases. All of these patients have a circulating C1EI of lower molecular weight than that of normal control subjects. When native C1EI was added to patient, but not control, plasmas, it was cleaved to a lower molecular weight fragment under conditions that allowed contact system activation. In a partially purified system, patient immunoglobulin preparations impaired stable complex formation between plasmin and C1EI and exaggerated the cleavage of the latter to a lower molecular weight fragment. We propose that the autoantibody does not interfere with the cleavage of the bait sequence of the inhibitor but reduces the availability of the reactive center of the molecule in a way that interferes with the irreversible inhibition of target enzymes. In this way unregulated activation of the kinin and complement pathways occurs, leading to disease manifestations via as yet uncharacterized mediators.