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1.
Fibronectin ("cold-insoluble globulin") has been suggested to play a role in cell-to-cell and cell-to-substratum adhesions. The 70-kilodalton terminal part of human fibronectin has recently been shown to bind to Staphylococcus aureus. In the present study, a fibronectin-binding protein was purified from sonicated S. aureus strain E2371 by affinity chromatography on fibronectin-Sepharose. The fibronectin-binding protein was isolated from an extract of sonicated S. aureus containing at least 57 different proteins as determined by crossed immunoelectrophoresis in antibodies to sonicated S. aureus. The fibronectin-binding protein was released from fibronectin-Sepharose by carbamide (8 M). No impurities in the final preparation could be detected when tested in crossed immunoelectrophoresis. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed two bands with relative molecular masses of 197,000 and 60,000, respectively. A complex between the purified S. aureus protein and fibronectin could be demonstrated by crossed immunoelectrophoresis both in monospecific antibodies against fibronectin and in S. aureus polyspecific antibody. 相似文献
2.
A protein (gal-FnBP), constructed by fusion of the genes encoding beta-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25% respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component. 相似文献
3.
Analysis of Ebh,a 1.1-megadalton cell wall-associated fibronectin-binding protein of Staphylococcus aureus
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In order for Staphylococcus aureus to adhere to host extracellular matrix (ECM) substrates, it elicits a wide range of surface proteins. We have characterized a novel approximately 1.1-MDa protein in S. aureus, termed Ebh (for ECM-binding protein homologue), which has homology to other ECM-binding proteins. Ebh consists of several domains, including a large central region with 44 imperfect repeats of 126 amino acids. Expression analysis revealed ebh to be growth phase regulated and repressed by agr. A fragment of the central repeat region of Ebh was cloned, overexpressed, and used in ligand-binding studies to determine Ebh function. The recombinant protein was found to specifically bind human fibronectin. Ebh is produced during human infection since serum samples taken from patients with confirmed S. aureus infections were found to contain anti-Ebh antibodies. Localization studies revealed Ebh to be cell envelope associated and is proposed to form a specialized surface structure involved in cellular adhesion. 相似文献
4.
Piroth L Que YA Widmer E Panchaud A Piu S Entenza JM Moreillon P 《Infection and immunity》2008,76(8):3824-3831
Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties—as well as elastin binding—and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by ≥10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies. 相似文献
5.
Staphylococcus epidermidis is a commensal of human skin and a leading cause of nosocomial bloodstream infections. Limited information is available about S. epidermidis proteins that are expressed upon transition to the bloodstream or those involved in host-pathogen interactions. A cell surface fraction from S. epidermidis 0-47 grown in rabbit serum to mimic environmental signals encountered during a bloodstream infection was separated by two-dimensional (2D) gel electrophoresis. Following 2D separation, the proteins were transferred to nitrocellulose and detected with either pooled sera generated in rabbits immunized with live S. epidermidis 0-47 or with biotin-labeled serum proteins eluted from the surface of bacteria grown in rabbit serum. Twenty-nine immunoreactive or serum binding proteins of S. epidermidis were identified by mass spectrometry. Twenty-seven of the corresponding genes were expressed in Escherichia coli, and the purified recombinant proteins were used to immunize mice. In a preliminary screen, 12 of the 27 recombinant proteins induced a response that reduced the number of bacteria recovered from the spleen or bloodstream of infected mice. In subsequent vaccination studies, 5 of the 12 proteins resulted in a statistically significant reduction in the number of bacteria. The identification of five candidate vaccine antigens from the initial screen of only 29 proteins demonstrates the utility of this approach. 相似文献
6.
Functional Studies of a Fibrinogen Binding Protein from Staphylococcus epidermidis 总被引:3,自引:0,他引:3
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Lei Pei Marco Palma Martin Nilsson Bengt Guss Jan-Ingmar Flock 《Infection and immunity》1999,67(9):4525-4530
A gene encoding a fibrinogen binding protein from Staphylococcus epidermidis was previously cloned, and the nucleotide sequence was determined. A portion of the gene encompassing the fibrinogen binding domain has now been subcloned in an expression-fusion vector. The fusion protein can bind to fibrinogen in a capture enzyme-linked immunosorbent assay and can be purified by fibrinogen affinity chromatography. This protein can completely inhibit the adherence of S. epidermidis to immobilized fibrinogen, suggesting that the adherence of S. epidermidis to fibrinogen is mainly due to this protein. Antibodies against this fibrinogen binding protein were also found to efficiently block the adherence of S. epidermidis to immobilized fibrinogen. Despite homology with clumping factors A and B from S. aureus (cell surface-associated proteins binding to fibrinogen), binding involved the beta chain of fibrinogen rather than the gamma chain, as in clumping factor A. 相似文献
7.
Increased virulence of a fibronectin-binding protein mutant of Staphylococcus aureus in a rat model of pneumonia
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Fibronectin-binding proteins mediate Staphylococcus aureus internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using S. aureus strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate S. aureus internalization into alveolar epithelial cells in vitro and that S. aureus internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of S. aureus-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation. S. aureus growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that S. aureus was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of S. aureus in pneumonia. 相似文献
8.
A deltalike toxin produced by a clinical isolate of Staphylococcus epidermidis was purified, and the amino acid sequence was determined. The toxin molecule consisted of 25 amino acid residues and shared a high degree of molecular homology with delta toxin purified from a Staphylococcus aureus human isolate. 相似文献
9.
Identification of antigenic components of Staphylococcus epidermidis expressed during human infection
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A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease. 相似文献
10.
Characterization and molecular cloning of a glutamyl endopeptidase from Staphylococcus epidermidis 总被引:1,自引:0,他引:1
Ohara-Nemoto Y Ikeda Y Kobayashi M Sasaki M Tajika S Kimura S 《Microbial pathogenesis》2002,33(1):33-41
A novel extracellular endopeptidase, designated GluSE, was purified from Staphylococcus epidermidis ATCC 14990 cultured by the dialysis membrane technique, and the structural gene (gseA) was cloned. GluSE was a 27kDa, glutamic acid-specific protease, and the optimal pH was 8.0. The proteolytic activity was specifically inhibited with diisopropyl fluorophosphate, indicating that it is a serine protease. The gseA encoded a single polypeptide of 282 amino acids with a deduced molecular weight of 30,809, in which the first 19 N-terminal amino acids completely matched the deduced sequence starting at Val-67, suggesting that GluSE is synthesized with a propeptide. The amino acid sequence of GluSE exhibited 50.5% identity to Staphylococcus aureus V8-protease (GluV8). Although GluSE lacks a C-terminal 12 repeats of the PBN/PBZ tripeptide of GluV8, a catalytic triad of His-117, Asp-159 and Ser-235 was conserved in GluSE. Southern hybridization analysis revealed that gseA exists as a single copy on the chromosomal DNA. The finding that production of GluSE was obviously observed in the adherent culture conditions of the dialysis membrane technique, but not in the planktonic culture conditions, strongly suggests that GluSE could be involved in an important etiologic process in S.epidermidis infection leading to multiple tissue damages. 相似文献
11.
Chiou JY Wang TM Chang CH Wu JJ Tsai WC 《Zhonghua Minguo wei sheng wu ji mian yi xue za zhi = Chinese journal of microbiology and immunology》1997,30(4):275-280
The importance of coagulase-negative staphylococci, especially Staphylococcus epidermidis in clinical and nosocomial infection are recognized increasingly in recent years. A rapid and accurate identification of S. epidermidis is therefore important and necessary. A new test, susceptibility to desferrioxamine, coupled with trehalose fermentation has been recommended for the identification of this organism. However, the medium and method used are different from what has been recommended by the NCCLS. To investigate the feasibility of using the desferrioxamine susceptibility test in conjunction with the routinely used disc agar diffusion test, we employed 111 staphylococcal strains (including 51 S. epidermidis isolates, 15 S. hominis and 45 other coagulase-negative staphylococci) as test organisms, and followed the procedures recommended by the NCCLS in which Mueller-Hinton agar and standard inoculum were used. Results indicated that all strains of S. epidermidis and S. hominis were susceptible to 1 mg desferrioxamine (the diameter of the inhibition zone were 28-37 mm). The minimum inhibitory concentrations of desferrioxamine to S. epidermidis and S. hominis isolates were determined to be 125 micrograms/ml. Further differentiation of S. hominis and S. epidermidis can be made by their ability to ferment trehalose, the former could while the latter could not. We conclude that the desferrioxamine susceptibility test of coagulase-negative staphylococci can be used in conjunction with the routine disc agar diffusion method. S. epidermidis can be identified rapidly and accurately by its susceptibility to 1 mg desferrioxamine and inability to ferment trehalose. 相似文献
12.
E. Carretto D. Barbarini I. Couto D. De Vitis P. Marone J. Verhoef H. De Lencastre S. Brisse 《Clinical microbiology and infection》2005,11(3):177-184
As routine identification of coagulase-negative staphylococci is problematic, the performance of automated ribotyping was evaluated for identification of coagulase-negative staphylococci other than Staphylococcus epidermidis. In total, 177 isolates were tested, comprising 149 isolates from blood samples, 15 isolates that were not identified by internal transcribed spacer (ITS)-PCR in a previous study, and 13 reference strains. The identification results were compared with those obtained by the API 20 Staph system, with standard phenotypic and molecular methods as reference. Most (n = 166; 93.8%) isolates were identified correctly by automated ribotyping. For 61 isolates, API 20 Staph and ribotyping were in agreement, but for 105 isolates, ribotyping provided correct identification and API 20 Staph did not. Four isolates not identified by automated ribotyping were recognised correctly by API 20 Staph. The remaining seven isolates could not be identified by either of the two methods. Automated ribotyping was able to distinguish Staphylococcus capitis reliably from Staphylococcus caprae. The results demonstrate the value of automated ribotyping for identification of coagulase-negative Staphylococcus (CoNS) isolates from human sources and may help to clarify the clinical relevance of CoNS species. In addition, automated ribotyping was able to detect polymorphisms that may be useful for epidemiological purposes within S. capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus simulans, S. caprae, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus sciuri, Staphylococcus pasteuri and Staphylococcus xylosus. 相似文献
13.
The fibrinogen binding protein of Staphylococcus epidermidis is a target for opsonic antibodies 总被引:1,自引:0,他引:1
Antibodies against the fibrinogen binding protein (Fbe) of Staphylococcus epidermidis significantly increased macrophage phagocytosis. Antibodies against autolysin E were opsonic but to a lesser extent. Antibodies against a novel, putatively surface-located antigen were unable to enhance phagocytosis. The severity of systemic infection of mice with S. epidermidis was reduced if the bacteria were preopsonized with anti-Fbe prior to administration. Fbe is thus a strong candidate for protein vaccination against S. epidermidis infection, and antibodies against Fbe can be used to prevent or treat infections caused by S. epidermidis. 相似文献
14.
Identification of a fibronectin-binding protein (GfbA) in pathogenic group G streptococci. 总被引:2,自引:3,他引:2
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Attachment to eukaryotic cell surfaces is an essential step in the establishment of colonization and infection by bacterial pathogens. This report examines the adherence capabilities of pathogenic group G streptococci and demonstrates that certain group G streptococcal clinical isolates express a fibronectin-binding protein. This protein, termed GfbA for group G streptococcal fibronectin-binding protein, mediates adherence to human skin fibroblasts (HSF). The gene encoding this protein, gfbA, was isolated, and the complete DNA sequence of gfbA was determined. From this sequence GfbA was predicted to be a 580-amino-acid protein (molecular weight = 64,979) with significant amino acid identity to the group A streptococcal fibronectin-binding proteins SfbI and protein F (PrtF) (76 and 78% identity, respectively). GfbA contains regions with notable identity to the fibronectin-binding repeat domains of PrtF. gfbA(+) strains were able to bind to HSF, and preincubation of the gfbA(+) strains with fibronectin blocked this adherence. In addition, gfbA(+) strains were able to bind radiolabeled fibronectin, and this binding was inhibited with addition of excess unlabeled fibronectin. gfbA-negative strains were not able to bind either the HSF or radiolabeled fibronectin. DNA homologous to gfbA was found in 36% of the group G streptococcal isolates examined. Since not all group G streptococcal strains examined contained gfbA, this suggests there might be other tissue-specific adherence molecules expressed by these pathogenic strains. 相似文献
15.
Jeong J Chang CL Park TS Lee SH Kim SR Jeong SH 《Journal of Korean medical science》2002,17(2):168-172
The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance. 相似文献
16.
Identification of an antigenic marker of slime production for Staphylococcus epidermidis. 总被引:3,自引:6,他引:3
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G D Christensen L P Barker T P Mawhinney L M Baddour W A Simpson 《Infection and immunity》1990,58(9):2906-2911
The pathogenic Staphylococcus epidermidis strain RP62A (ATCC 35984) adheres to smooth surfaces by forming a tenacious bacterial film known as slime. The mechanism of slime production is not known; however, workers in the laboratory of G. Pier (Harvard Medical School, Boston, Mass.) have isolated from RP62A a galactose-rich capsular polysaccharide adhesin (CPA) which mediates the attachment of the organism to smooth surfaces. We have obtained two daughter strains from RP62A that no longer produce slime. One daughter strain, H4A, was obtained by selection for a spontaneous variant; the other strain, HAM892, was obtained by treating growing cultures of RP62A with acriflavin. Using an antiserum generated against whole cells of RP62A, we have examined lysozyme-lysostaphin digests of RP62A, H4A, and HAM892 by double immunodiffusion. The two strains that no longer produced slime no longer produced a particular antigen, which we refer to as the slime-associated antigen (SAA). SAA was also produced by unrelated strains of slime-producing S. epidermidis. SAA was heat and protease stable, had a molecular weight of greater than 50,000, and could be partially purified by chromatographing trypsin-digested material over a Sephadex G-200 column. Chemical analysis of partially purified SAA by gas-liquid chromatography found SAA to be glucose rich (59%) and galactose poor (1.4%). This analysis chemically distinguished SAA from CPA. When tested together by double immunodiffusion with anti-RP62A and anti-CPA antisera, partially purified SAA did not cross-react with CPA. Kinetic studies suggested that SAA is a marker for surface accumulation whereas CPA mediates initial adherence. 相似文献
17.
Characterization of a sar Homolog of Staphylococcus epidermidis 总被引:1,自引:0,他引:1
18.
Rapid identification of Staphylococcus epidermidis 总被引:1,自引:0,他引:1
G L Woods G S Hall I Rutherford K Pratt C C Knapp 《American journal of clinical pathology》1987,87(4):525-528
A panel of Minitek sugar disks, consisting of trehalose, mannitol, xylose, and sucrose, was evaluated for its ability to identify blood culture isolates of Staphylococcus epidermidis (SE). Using a heavy suspension of organism in Mueller-Hinton broth, 50 microL was pipetted onto each disk in wells of a flat-bottomed microtiter tray. The tray was covered, incubated in a moist chamber in non-CO2 at 35 degrees C, and examined after 5 and 24 hours. A color change of yellow or orange was positive; no color change (red) was negative. Expected reactions for SE were as follows: negative trehalose, mannitol, and xylose; positive, sucrose. On evaluation of 227 coagulase-negative staphylococci (CNS) at 5 and 24 hours, the panel had a sensitivity of 94 and 96%, specificity of 92 and 89%, predictive value of positive tests of 97 and 96%, and predictive value of negative tests of 84 and 87%. This panel offered an inexpensive and convenient method for differentiating SE from the other CNS in five hours. 相似文献
19.
An elastase of Staphylococcus epidermidis was purified by ion exchange chromatography on CM-Sepharose and characterised. Its M(r) is c. 21 kDa, its optimal temperature for activity is 42 degrees C and the pH optimum is 6.8. The enzyme is activated by cysteine and other SH-donators and inhibited by L-trans-epoxy-succinylleucylamido-(4-guanidino)butane (E64), an inhibitor of cysteine proteases, but not by 3,4-dichloroisocoumarin (3,4-DCI), an inhibitor of serine proteases. This finding suggests that the elastase of S. epidermidis is a cysteine protease. Because S. epidermidis elastase degrades human sIgA, IgM, serum albumin, fibrinogen, and fibronectin, this enzyme may be regarded as a virulence factor. 相似文献
20.
Opsonic requirements of Staphylococcus epidermidis 总被引:3,自引:0,他引:3
The opsonic requirements of 18 strains of Staphylococcus epidermidis were compared in pooled normal human serum and in peritoneal dialysate from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A serum concentration of 2.5% gave optimal opsonisation. The opsonisation of all strains was antibody- and complement-dependent, and there were no significant differences in the pattern of their opsonic requirements. Peritoneal-dialysis effluent from uninfected patients was a poor source of opsonins because of the low levels of immunoglobulin G and of the C3 component of complement it contained. Growth of S. epidermidis in peritoneal-dialysis effluent rather than in broth did not alter its opsonic requirements. Strains from patients undergoing CAPD and suffering from peritonitis were not more resistant to opsonisation and phagocytic killing than those from other sources. 相似文献