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M Caparon

Publications and source records attributed to M Caparon.

16 recordsLinked to original sources
PubMed2001

Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in gram-positive bacteria.

Type III secretion for injection of effector proteins into host cells has not been described for Gram-positive bacteria despite their importance in disease. Here, we describe an injection pathway for the Gram-positive pathogen Streptococcus pyogenes that utilizes streptolysin O (SLO), a cholesterol-dependent cytolysin. The data support a model in which an effector is translocated through the SLO pore by a polarized process. The effector, SPN (S. pyogenes NAD-glycohydrolase), is capable of producing the potent second messenger cyclic ADP-ribose, and SLO and SPN act synergistically to trigger cytotoxicity. These data provide a novel paradigm for the function of the cholesterol-dependent cytolysin family and its wide distribution suggests that cytolysin-mediated translocation (CMT) may be the equivalent of type III secretion for Gram-positive pathogens.

Adenosine Diphosphate Ribose↗
PubMed1998

Streptolysin O and adherence synergistically modulate proinflammatory responses of keratinocytes to group A streptococci.

In contrast to a mutant adhesin-deficient Streptococcus pyogenes (group A streptococcus), its isogenic parental strain binds to human keratinocytes and promotes a vigorous proinflammatory response, characterized by enhanced expression of several cytokines, a more rapid release of prostaglandin E2 (PGE2) and damage to keratinocyte membranes. However, adherence alone is not sufficient to induce these responses. In this study, we have begun to examine the contribution of other streptococcal products in interactions with keratinocytes by the construction and evaluation of mutants deficient in expression of the secreted pore-forming haemolysin, streptolysin O (SLO). Inactivation of SLO did not prevent the streptococci from adhering to cultured HaCaT keratinocytes or from expressing an unrelated second streptococcal haemolysin, streptolysin S, during infection of keratinocytes. As measured by a quantitative reverse transcriptase polymerase chain reaction (PCR) assay, inactivation of SLO also did not have a marked effect on the expression of interleukin 1alpha (IL-1alpha) during infection. However, the lack of the ability to produce SLO was associated with a considerable reduction in expression of IL-1beta, IL-6 and IL-8 by infected keratinocytes. Measurement of the release of PGE2 by an enzyme-linked immunosorbent assay demonstrated that the SLO-deficient mutants were also not capable of promoting the rapid high level of PGE2 release characteristic of the adherent SLO-producing parental strain. Finally, analyses using the fluorescent probe ethidium homodimer-1 and measurements of release of keratinocyte lactate dehydrogenase indicated that the failure of the SLO-deficient mutants to induce responses was associated with the failure of these mutants to damage the integrity of the keratinocyte membrane. These data implicate SLO as a factor that acts synergistically with an adhesin to modulate the signalling responses of keratinocytes during infection.

Bacterial Adhesion↗
PubMed1997

A matrix form of fibronectin mediates enhanced binding of Streptococcus pyogenes to host tissue.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) binds to fibronectin via protein F. In this study, we have investigated the binding properties of protein F to various multimeric tissue forms of fibronectin that appear on cell surfaces and in the extracellular matrix. We show that binding of S. pyogenes through protein F is more efficient to an in vitro-derived polymerized form of fibronectin (superfibronectin) than to soluble fibronectin immobilized in a solid phase. In addition, Chinese hamster ovary cells overexpressing the alpha5beta1 integrin produced an increased amount of a fibronectin matrix and consequently bound a higher number of S. pyogenes cells. Inhibition and direct binding assays using purified proteins demonstrated that binding to a fibronectin matrix involved both domains of protein F (UR and RD2) that have previously been implicated in interactions with fibronectin. Using intact S. pyogenes bacteria in which various domains of protein F were expressed as hybrids with the surface-exposed region of an unrelated protein, we revealed that, in contrast to the predominantly UR-mediated binding to soluble fibronectin, the maximal binding to the fibronectin matrix required RD2 in addition to UR. Since in some infections S. pyogenes may initially encounter a matrix form of fibronectin, these results suggest that UR and RD2 may be important for the initiation of streptococcal infectious processes.

Adhesins, Bacterial↗
PubMed1997

Attenuation of IL-5-mediated signal transduction, eosinophil survival, and inflammatory mediator release by a soluble human IL-5 receptor.

A soluble form of the human IL-5R alpha-chain (IL-5Ra) that contains the extracellular IL-5 binding domain has been evaluated for its effect on IL-5 binding to and activation of human eosinophils and basophils. The truncated receptor was expressed in Escherichia coli and recovered in biologically active form following renaturation and anion exchange chromatography. The soluble receptor formed a 1/1 complex with IL-5 in solution and bound IL-5 with affinity comparable to that of cell-associated IL-5Ra. Soluble IL-5Ra also competed with IL-5 for binding to the native alpha beta IL-5R on human cells and inhibited IL-5-mediated receptor activation and inflammatory mediator production. In this regard, the soluble receptor prevented IL-5-induced tyrosine phosphorylation of JAK2 kinase and IL-5R beta-chain and inhibited IL-5 priming of leukotriene C4 release by human basophils. However, the E. coli-derived receptor failed to inhibit IL-5 in longer term assays, including eosinophil survival and TF-1 cell proliferation, possibly due to its propensity to aggregate in a time- and temperature-dependent manner. In contrast, we observed that a soluble IL-5Ra derived from baculovirus-infected cells was less prone to aggregate and effectively antagonized IL-5-induced cell proliferation and survival. These findings indicate that the extracellular portion of the human IL-5Ra chain can prevent association of IL-5 with cell surface receptors and can attenuate signal transduction, mediator release, and survival of inflammatory cells. As such, soluble IL-5R may be useful in treating diseases such as human asthma, in which pulmonary injury is associated with the activity of IL-5R-bearing cells.

Basophils↗
PubMed1997

Polymerase chain reaction for Streptococcus pyogenes used to evaluate an optical immunoassay for the detection of group A streptococci in children with pharyngitis.

BACKGROUND: In evaluations of sensitive rapid tests for group A streptococci such as the optical immunoassay (OIA), some samples are positive by the antigen test but negative by culture. A method is needed for resolving these discrepant results. OBJECTIVE: To develop a PCR-based assay to detect group A streptococci and to use it to establish a reference standard for evaluating an OIA for group A streptococcal antigen. METHODS: A PCR assay that detects a segment of the MF gene of Streptococcus pyogenes was developed for the detection of group A streptococci in throat swabs. Paired swabs were obtained from 200 children with symptomatic pharyngitis and used to perform OIA, agar culture, broth-enhanced culture and PCR. As a reference standard any patient with group A streptococci detected by either culture or PCR was considered to be truly positive. RESULTS: In comparison to agar and broth-enhanced culture procedures, OIA had sensitivities of 82 and 80% and specificities of 87 and 89%, respectively. Eight (44%) of 18 samples that were positive by OIA but negative by culture were positive for group A streptococci by PCR. Compared with the reference standard, sensitivities were OIA 76%, agar culture 79%, broth-enhanced culture 86% and PCR 96%. The specificity of OIA was 92%. CONCLUSIONS: PCR can be used to establish a reference standard for evaluating rapid tests for group A streptococci. With this reference standard OIA was nearly as sensitive as but less specific than agar culture for detection of group A streptococci. Maximum detection requires use of both tests.

Antigens, Bacterial↗
PubMed1997

Keratinocyte proinflammatory responses to adherent and nonadherent group A streptococci.

The gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of a wide variety of suppurative infections of cutaneous tissues. Previous analyses have demonstrated that the M protein of S. pyogenes is an adhesin that directs the attachment of the streptococcus to keratinocytes in the skin. In this study, we have examined keratinocyte function in response to S. pyogenes and found that adherent versus nonadherent streptococci promote distinct patterns of expression of several proinflammatory molecules and keratinocyte cell fate. When analyzed by a quantitative reverse transcriptase PCR method, infection of cultured HaCaT keratinocytes with adherent, but not nonadherent, streptococci resulted in increased expression of mRNA for the cytokines interleukin-1alpha (IL-1alpha), IL-1beta, and IL-8 but neither infection induced expression of tumor necrosis factor alpha. In contrast, both adherent and nonadherent S. pyogenes induced expression of IL-6 and each promoted synthesis and release of prostaglandin E2 (PGE2). However, considerably greater levels of IL-6 expression were stimulated by adherent streptococci relative to nonadherent streptococci and the kinetics of PGE2 release in response to nonadherent streptococci was delayed compared to the response to adherent streptococci. Staining with the fluorescent probe ethidium homodimer-1 revealed that keratinocyte membranes were rapidly damaged upon infection with adherent streptococci but were not damaged by nonadherent streptococci. Finally, treatments which inhibited streptococcal metabolism completely blocked the ability of adherent streptococci to elicit responses. These data suggest that expression of an adhesin is a strategy used by S. pyogenes to modulate keratinocyte responses during infection of the skin and implicate additional streptococcal products in these signaling interactions.

Bacterial Adhesion↗
PubMed1996

A peptide isolated by phage display binds to ICAM-1 and inhibits binding to LFA-1.

A mixed phage library containing random peptides from four to eight residues in length flanked by cysteine residues was screened using a recombinant soluble, form of human ICAM-1, which included residues 1-453, (ICAM-1(1-453)). Phage bound to immobilized ICAM-1(1-453) were eluted by three methods: (1) soluble ICAM-1(1-453), (2) neutralizing murine monoclonal antibody, (anti-ICAM-1, M174F5B7), (3) acidic conditions. After three rounds of binding and elution, a single, unique ICAM-1 binding phage bearing the peptide EWCEYLGGYLRCYA was isolated; the identical phage was selected with each method of elution. Attempts to isolate phage from non-constrained (i.e., not containing cysteines) libraries did not yield a phage that bound to ICAM-1. Phage displaying EWCEYLGGYLRCYA bound to immobilized ICAM-1(1-453) and to ICAM-1(1-185), a recombinant ICAM-1, which contains only the two amino-terminal immunoglobulin domains residing within residues 1-185. This is the region of the ICAM-1 that is bound by LFA-1. The phage did not bind to proteins other than ICAM-1. The phage bound to two ICAM-1 mutants, which contained amino acid substitutions that dramatically decreased or eliminated the binding to LFA-1. Studies were also performed with the corresponding synthetic peptide. The linear form of the synthetic EWCEYLGGYLRCYA peptide was found to inhibit LFA-1 binding to immobilized ICAM-1(1-453) in a protein-protein binding assay. By contrast, the disulfide, cyclized, form of the peptide was inactive. The EWCEYL portion of the sequence is homologous to the EWPEYL sequence found within rhinovirus coat protein 14, a nonintegrin protein that binds to ICAM-1. Taken together, the results suggests that the EWCEYLGGYLRCYA sequence is capable to binding to immobilized ICAM-1. Phage display appears to represent a new approach for the identification of peptides that interfere with ICAM-1 binding to beta 2 integrins.

Bacteriophages↗
PubMed1996

An oxygen-induced but protein F-independent fibronectin-binding pathway in Streptococcus pyogenes.

Protein F is an important fibronectin-binding adhesin of Streptococcus pyogenes (group A streptococcus). However, all previous analyses of protein F have been conducted in a mutant strain which expresses protein F under anaerobic conditions nonpermissive for expression in other strains. In this study, we have examined the fibronectin-binding properties of several protein F-deficient mutants cultured under aerobic conditions and have identified a second pathway for binding fibronectin. Unlike the case with protein F, exposure to an aerobic environment does not induce transcription of a new gene product. Rather, O2 is apparently required for the modification of a protease-resistant cell surface component into a binding-component form. Modification occurred preferentially at a pH of 6.0 or less, and the binding of the modified component to fibronectin required Zn2+. The oxidizing agent Fe(CN)6 could be substituted for O2 and stimulated expression of binding activity under O2-limiting conditions. Streptococcal fibronectin binding mediated by this pathway but not by protein F could be inhibited by laminin and by streptococcal lipoteichoic acid, a molecule previously implicated as the streptococcal adhesin for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism to basement membrane. By using differential inhibition, analyses of binding to non-protein F mutant strains demonstrated that the total level of fibronectin bound under aerobic conditions reflects contributions from both pathways. Because of its dependence on Zn2+, an oxidant, and pH, this binding activity has been designated the ZOP binding pathway.

Adhesins, Bacterial↗
PubMed1995

Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

Antigens, Bacterial↗
PubMed1995

Regulation of host cell recognition in Streptococcus pyogenes.

Protein F, a fibronectin-binding protein, and the M protein of Streptococcus pyogenes both play important roles in directing adherence to different populations of host cells in the skin. Expression of both proteins is regulated in response to alterations in atmosphere. Transcriptional control of mry, a positive-acting regulator of expression of the gene which encodes the M protein (emm) in response, to elevated levels of CO2, is a mechanism for control of emm expression in response to atmosphere. Expression of protein F is controlled at the level of transcription in response to the concentration of O2, and its expression is stimulated in the presence of superoxide. Further support for a role of superoxide in regulation of prtF expression comes from the observation that an S. pyogenes mutant which contains an insertionally inactivated gene for superoxide dismutase (sod) becomes hypersensitive to superoxide and will express prtF constitutively. A second strain also demonstrates constitutive expression of prtF but contains a functional sod. Complementation analyses in this strain using a prtF allele cloned from a regulating host and a novel method for shuttle mutagenesis which utilized the transposon mini-gamma delta have been used to identify rofA, a positive-acting regulator of prtF expression. A model for the role of the surface proteins, F and M and their regulatory genes mry and rofA in streptococcal infections of the skin will be discussed.

Antigens, Bacterial↗
PubMed1993

Adherence and fibronectin binding are environmentally regulated in the group A streptococci.

The ability of the pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) to bind fibronectin and adhere to respiratory epithelial cells is dependent on a surface protein called protein F. In this study, we have examined the regulation of expression of protein F and have shown that it is environmentally regulated in response to alterations in atmosphere. In six recent clinical isolates expression of protein F was repressed during growth under reduced concentrations of O2. Expression in an anaerobic environment was induced by both superoxide-generating and redox-altering reagents. However, regulation did not involve mry, a gene that controls expression of several streptococcal surface proteins. Protein F was constitutively expressed in one of two laboratory-passaged strains analysed, and in a complementation analysis using an allele of the gene that encodes protein F (prtF) cloned from a regulated strain and expressed in a constitutive strain, the constitutive phenotype was shown to be dominant in trans. Regulation, as monitored by fusion of prtF to a promoterless chloramphenicol acetyltransferase gene, involved transcriptional control. Environmentally induced alterations in protein F expression affected the ability of the bacterium to adhere to epithelial cells, which suggests that the ability to regulate expression of protein F may be important during infection.

Bacterial Adhesion↗
PubMed1992

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

Animals↗
PubMedDate not supplied

Macrophage toxicity and complement sensitivity of virulent and avirulent strains of Legionella pneumophila.

Virulent strains of Legionella pneumophila that are preincubated with specific antiserum are cytotoxic for alveolar macrophages when added at a multiplicity of infection of 100 L. pneumophila per macrophage. Under the same conditions, avirulent strains exhibit little cytotoxic activity. Virulent strains are also resistant to the bactericidal activity of normal human serum and fail to bind complement components on the cell surface. In contrast, avirulent strains are serum-sensitive and both C3 and C9 are deposited on the surface. Results of experiments suggest that the shift from virulence to avirulence of L. pneumophila may be the result of the selection of a small number of avirulent cells present in the population of a virulent culture.

Animals↗