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E H Beachey

Publications and source records attributed to E H Beachey.

At least 37 records · Page 2Linked to original sources
PubMed1989

Fibrinogen binding and resistance to phagocytosis of Streptococcus sanguis expressing cloned M protein of Streptococcus pyogenes.

The biological properties of Streptococcus pyogenes M protein cloned and expressed in S. sanguis were investigated. The spm-5 gene previously cloned into Escherichia coli was subcloned into the E. coli-S. sanguis shuttle plasmid pVA838 to produce a newly constructed plasmid, pBK100. Cells of S. sanguis transformed with pBK100 expressed 53-, 55-, and 58-kilodalton polypeptides reacting with type 5 M protein antiserum in immunoblots. The M protein was expressed on the surface of S. sanguis cells as shown by the capacity of the intact cells to (i) inhibit the reactivity of anti-type 5 antibodies with purified M protein as demonstrated by enzyme-linked immunosorbent assay; (ii) inhibit the opsonization by M5 antisera of type 5 S. pyogenes; (iii) express M-protein-like fibrils on the surface of the organisms that react with M5 antisera as revealed by immunoelectron microscopy; (iv) bind plasma fibrinogen and, as a consequence, resist phagocytosis by human blood neutrophils; and (v) be rendered susceptible to phagocytosis by opsonic M5 antisera. These results provide additional evidence that streptococcal M proteins bind host proteins as a ploy to evade host defense mechanisms.

Antigens, Bacterial↗
PubMed1989

Ultrastructural localization of the fibrinogen-binding domain of streptococcal M protein.

Binding of fibrinogen to the M protein located on the surface fibrillae of group A streptococci impedes deposition of complement and thus contributes to the virulence of these organisms. We investigated this binding by electron microscopy using postembedding immunogold labeling. Both fibrinogen and its D fragment formed a distinct dense layer in the surface fibrillae, separated by 10 nm from the compact part of the cell wall. Labeling the sections with anti-fibrinogen or anti-fragment D showed that the fibrinogen-binding region lay within a 25-nm segment of the fibrillae beginning approximately 30 nm from the inner surface of the cell wall. The outer surface of the fibrinogen layer could be labeled with antibody to the amino-terminal half of type 24 M protein, indicating that the fibrillar tips remained exposed after fibrinogen binding. The degree of labeling with anti-fibrinogen, determined by gold particle counting, was the same whether the bacterial cells had been incubated with purified fibrinogen or whole plasma. These results indicate that the fibrinogen-binding region lies in the distal (amino-terminal) half of the M protein molecule but excludes the most distal portion, which is the site of epitopes that interact with opsonic anti-M antibody, and that plasma proteins other than fibrinogen, a number of which are known to bind to group A streptococci, do not interfere with fibrinogen binding.

Antigens, Bacterial↗
PubMed1989

Vimentin-cross-reactive epitope of type 12 streptococcal M protein.

The NH2-terminal amino acid sequence of type 12 M protein was determined by automated Edman degradation of a 38-kilodalton polypeptide fragment purified from a limited pepsin digest of intact type 12 streptococci. The sequence of the first 13 amino acid residues of the polypeptide confirmed that predicted by the nucleotide sequence of the mature type 12 M protein. A chemically synthesized peptide copying the NH2-terminal 25 residues, SM12(1-25)C, evoked opsonic antibodies against type 12 streptococci as well as renal glomerular cross-reactive antibodies. The serum from one of six rabbits reacted in immunofluorescence tests with human glomeruli in a mesangial staining pattern. The cross-reactive antibodies were completely inhibited by the immunizing peptide and absorption with type 12 streptococci. Subpeptides of the 25-residue synthetic peptide were without inhibitory effect, suggesting that the cross-reactive antibodies are directed against a conformational epitope of SM12(1-25)C. Anti-SM12(1-25)C antisera reacted specifically with the intermediate filament protein vimentin extracted from mesangial cells. None of the cross-reactions of anti-SM12(1-25)C were inhibited by a synthetic peptide SM1(1-26)C of type 1 M protein, which was previously shown to share a cross-reactive epitope with vimentin. These results indicate that type 12 M protein contains at least one vimentin cross-reactive epitope that is clearly distinct from the tetrapeptide epitope shared with vimentin by type 1 M protein.

Amino Acid Sequence↗
PubMed1989

Bacterial adherence of group A streptococci to mucosal surfaces.

It is now recognized that bacteria bind to and colonize mucosal surfaces in a highly selective manner. After the organisms penetrate the nonspecific mechanical and cleansing forces, ligands (or adhesins) on the surface of the bacteria interact in a lock-and-key (or induced fit) fashion with complementary receptors on mucosal surfaces of the host. The adhesins are usually composed of proteins in the form of fimbriae or fibrillae and the receptors of glycolipids or glycoproteins. In group-A streptococci the adhesin, lipoteichoic acid (LTA), is anchored to a protein(s) on the surface of the bacterial cells and interacts through its lipid moiety with fibronectin molecules deposited on and bound to the epithelial cells. In an attempt to locate the region of fibronectin recognized by LTA and group-A streptococci, fibronectin was cleaved with thermolysin and the fragment mixture absorbed with Staphylococcus aureus or Streptococcus pyogenes. Staphylococci adsorbed several high molecular weight fragments as well as a 28- and a 23-kdalton fragment, whereas S. pyogenes cells absorbed only the 28-kdalton fragment completely. The adsorbtion of the fragments by S. pyogenes was blocked by LTA. Antibodies raised against a synthetic peptide copying the NH2 terminus of fibronectin reacted in a Western blot with the 28-kdalton fragment, indicating that S. pyogenes and its LTA react with the NH2-terminal region of fibronectin at a site distinct from that of S. aureus. Our findings are consistent with the idea that LTA mediates the attachment of group-A streptococci to fatty acid-binding sites of fibronectin deposited on mucosal epithelial cells.

Bacterial Adhesion↗
PubMed1988

Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae.

A variety of genera and species of the family Enterobacteriaceae bear surface fimbriae that enable them to bind to D-mannose residues on eukaryotic cells. Until recently, it was thought that the D-mannose binding site was located in the major structural subunit (FimA), of relative molecular mass (Mr) 17,000 (17 K), of these organelles in Escherichia coli. New evidence indicates that this binding site resides instead in a minor protein Mr 28-31 K (FimH) located at the tips and at long intervals along the length of the fimbriae, and is reminiscent of the minor tip adhesion proteins of pyelonephritis-associated pili (Pap) and S fimbriae. In contrast to the antigenic heterogeneity of the major FimA subunit, the antigenic structure of FimH is conserved among different strains of E. coli. Here, we report an even broader conservation of this minor adhesion protein extending to other genera and species of type 1 fimbriated Enterobacteriaceae. Our results may have implications for the development of broadly protective vaccines against Gram-negative bacillary infections in animals and perhaps in man.

Antibodies, Monoclonal↗
PubMed1988

Protective immunity evoked by locally administered group A streptococcal vaccines in mice.

The present studies were undertaken to determine the pathogenicity of group A streptococci introduced intranasally (i.n.) into mice in an attempt to mimic mucosal infections in humans and to determine the efficacy of streptococcal vaccines administered via the mucosal route. The LD50 of type 24 streptococci (M24 strep) administered i.n. was 3 x 10(4) CFU. Throat cultures were performed in M24 strep-inoculated mice. Of 11 mice that died, 9 had positive throat cultures 3 or 4 days after i.n. challenge, and of 9 mice that survived, only 1 had a positive throat culture, indicating an association between mucosal infection and death. Postmortem examination performed on 35 mice that died after i.n. challenge showed that all had evidence of disseminated infections, and group A streptococci were recovered from the cervical lymph nodes, blood, spleen, liver, and brain. To determine vaccine efficacy, heat-killed M24 strep or pep M24 were administered i.n. to groups of mice. Whole, heat-killed streptococci and pep M24 administered locally protected mice against death from i.n. challenge infections with homologous M24 strep. The whole cell vaccine also protected against i.n. challenge infections with heterologous type 6 streptococci. Our data suggest that streptococcal vaccines administered locally evoke protective immunity against streptococcal infections.

Administration, Intranasal↗
PubMed1988

Protective immunity evoked by oral administration of attenuated aroA Salmonella typhimurium expressing cloned streptococcal M protein.

Attenuated strains of Salmonella have been used effectively as vaccines against typhoid fever. We have investigated the use of such strains to deliver cloned antiphagocytic virulence determinants of unrelated bacteria. The aroA strain of S. typhimurium SL3261 was transformed with a low-copy plasmid vector pMK207, which contains the cloned gene spm5 encoding streptococcal M protein, the major virulence factor of these organisms. The transformed SL3261 expressed type 5 M protein in the cytoplasmic fraction, and when fed orally to BALB/c mice, evoked both serum and salivary IgA, IgG, and IgM antibodies directed against type 5 M protein. The orally immunized mice were completely protected against both intranasal and intraperitoneal challenge infections with virulent S. typhimurium SL1344 or M5 streptococci. These studies provide evidence that an attenuated strain of Salmonella can be used effectively as a general vaccine vehicle to deliver antiphagocytic virulence determinants of unrelated bacteria.

Animals↗
PubMed1988

Protective and heart-crossreactive epitopes located within the NH2 terminus of type 19 streptococcal M protein.

M protein was purified to homogeneity from limited pepsin digests of intact type 19 streptococci (pep M19). The purified pep M19 when emulsified in CFA and injected into rabbits evoked type-specific and crossreactive opsonic antibodies, as well as heart-crossreactive antibodies. The NH2-terminal primary structure of pep M19 was determined and a peptide copying the first 24 amino acids [SM19(1-24)C] was chemically synthesized. Rabbits that were immunized with the unconjugated peptide developed antibodies that recognized the native pep M19, as determined by ELISA, and opsonic antibodies against type 19 streptococci, as determined by in vitro opsonophagocytosis tests. The synthetic peptide also evoked antibodies that crossreacted with a 60-kD sarcolemmal membrane protein of human myocardium. By using overlapping synthetic subpeptides as immunoinhibitors, the opsonic and heart-crossreactive epitopes of SM19(1-24)C were localized to SM19(11-24)C. Our data confirm the presence of heart-crossreactive epitopes within the primary structure of pep M19 and show that these potentially harmful autoimmune epitopes may be located in the NH2-terminal regions of certain M proteins. We conclude that continued efforts to identify the primary structures of protective and heart-crossreactive epitopes will be necessary to elucidate the pathogenesis of acute rheumatic heart disease and to develop safe and effective streptococcal vaccines.

Amino Acid Sequence↗
PubMed1988

Comparison of the leader sequences of four group A streptococcal M protein genes.

The 5' portions and flanking sequences of genes encoding types 1, 12, 24, and 6 M proteins were compared. Although the DNA sequences encoding the amino-termini of the mature M proteins had no obvious similarity, upstream sequences, and those encoding the signal peptides (leader sequences) of the four M protein genes had considerable similarity. In general, the 5' ends of all the leader sequences were more conserved than the 3' ends, although the M6 and M24 leader sequences had identical 3' ends. Sequence similarity among the deduced amino acid sequences of the four signal peptides was more extensive than the corresponding DNA sequences. We found that strict DNA similarity among all four sequences extended only to the ends of the hydrophilic amino-terminal regions of the signal peptides, but that amino acid sequence conservation continued to the ends of the respective hydrophobic cores. With the exception of the M6 and M24 sequences, the regions adjacent to the signal peptidase cleavage sites were highly variable.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes.

The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells. Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors. This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae. Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose. The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E. coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner. The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units. Isolated type 1 fimbriae inhibited the binding of fimbriated E. coli to purified receptor in a dose- and time-related fashion. The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.

Animals↗
PubMed1988

Influence of helical organization on immunogenicity and antigenicity of synthetic peptides.

Using cooperative effects of different peptide structures synthesized in tandem, we have induced an alpha-helical structure in water solution on a peptide which, alone, is unorganized. This structure is particularly relevant in this case as the selected model protein (type 24 M protein of Streptococcus pyogenes) is an extended coiled-coil system. We were thus able to assess the importance of organization or unorganization of a unique amino acid sequence with regards to its immunogenicity and antigenicity. Although in a classical manner, antibodies cross-reacting with the protein can be obtained with the short, unorganized peptide, we demonstrate that conformation-specific antibodies are raised when longer, organized peptides are used as immunogens.

Animals↗
PubMed1988

A sandwich adhesion on Streptococcus pneumoniae attaching to human oropharyngeal epithelial cells in vitro.

Streptococcus pneumoniae attach to human pharyngeal epithelial cells through the specific interaction of bacterial surface adhesins with glycoconjugate receptors. The present study defines the adhesin as a molecule bridging between an anchoring site in the bacterial cell wall and the epithelial cell receptor. The nature of the adhesin was defined in three ways: First, the attachment of whole bacteria was reduced by trypsin, periodate and heat. Second, heat treatment of whole bacteria was shown to release material, which was able to reconstitute the adherence. The heat extract bound to epithelial cells, as shown by fluorescence labelling, and agglutinated latex beads covalently coupled with receptor oligosaccharide. Active material could be extracted by heat from both high and low adhering strains, but could reconstitute only attaching strains. Third, the bacterial component binding the adhesin was localized to protoplasts and cell wall fractions obtained by mechanical or deoxycholate induced lysis of pneumococci. Isolated pneumococcal surface components, which did not inhibit attachment, included peptidoglycan, C polysaccharide, Forssmann antigen, capsular polysaccharide and a phenol extract produced in analogy to streptococcal lipoteichoic acid. The procedure used to extract the adhesin was previously used to prepare the competence factor. The competence deficient mutant RA7- attached poorly compared to the competent R6 parent. The possible relatedness of attachment to competence is discussed.

Adhesins, Bacterial↗
PubMed1988

Protective and autoimmune epitopes of streptococcal M proteins.

Several rheumatogenic serotypes of streptococcal M protein have been shown to contain both protective and cardiac tissue crossreactive epitopes. By synthesizing peptides copying different regions of M protein polypeptides, we were able to localize the protective and heart crossreactive epitopes. Some epitopes are only opsonic, some are only crossreactive, whereas others are both opsonic and tissue crossreactive. Multivalency of vaccines can be obtained by synthesizing protective peptides of one M serotype in tandem with protective peptides of other M serotypes. Such hybrid peptides evoke protective immune responses against the related streptococci without evoking tissue crossreactive immunity.

Animals↗
PubMed1988

Renal autoimmune epitope of group A streptococci specified by M protein tetrapeptide Ile-Arg-Leu-Arg.

The renal glomerular cross-reactivity of the amino-terminal region of type 1 streptococcal M protein was investigated. Antisera raised in rabbits against a synthetic peptide representing residues 1-26 and a peptide from which residues 20-22 had been omitted during synthesis were capable not only of opsonizing type 1 streptococci but also of reacting in immunofluorescence tests with human renal glomeruli. The cross-reactions were completely inhibited by the immunizing peptides. By using additional synthetic peptides in these inhibition studies, the glomerular cross-reactive epitope was localized to a tetrapeptide sequence Ile-Arg-Leu-Arg at positions 23-26. A number of synthetic M1 peptides containing the tetrapeptide sequence were inhibitory, whereas the M1 peptides lacking the sequence or unrelated tetrapeptides Arg-Gly-Asp-Ser or Arg-Gly-Phe-Ser were without effect. Furthermore, Ile-Arg-Leu-Arg affinity-purified antibodies reacted with renal glomeruli, and the reactivity was inhibited by the tetrapeptide as well as by type 1 M protein. These results indicate that a renal glomerular autoimmune epitope resides in a tetrapeptide Ile-Arg-Leu-Arg near the amino terminus of type 1 streptococcal M protein.

Amino Acid Sequence↗
PubMed1988

Influence of berberine sulfate on synthesis and expression of Pap fimbrial adhesin in uropathogenic Escherichia coli.

We investigated the influence of berberine sulfate, an ancient Chinese antibiotic, upon the adhesion of uropathogenic Escherichia coli to erythrocytes and epithelial cells. Although berberine sulfate in increasing concentrations had no effect on bacterial growth or on the synthesis of major outer membrane proteins of the E. coli organisms, it increasingly blocked adhesion. The decreased adhesion was accompanied by a reduction in the synthesis of fimbrial subunits and in the expression of assembled fimbriae. These results suggest that the anti-infectious activity of berberine sulfate in E. coli-induced urinary tract infections may be mediated by the selective suppression of the synthesis and assembly of fimbriae by uropathogenic organisms.

Adhesins, Escherichia coli↗
PubMed1988

Berberine sulfate blocks adherence of Streptococcus pyogenes to epithelial cells, fibronectin, and hexadecane.

Berberine sulfate is an alkaloid extracted from the roots and bark of various plants and possesses antibacterial, antifungal, and antiprotozoal activities. Most studies have focused on the bacteriostatic or bactericidal activities of this compound. In this study, we report that berberine sulfate is bacteriostatic for streptococci and that sub-MICs of berberine blocked the adherence of streptococci to host cells, immobilized fibronectin, and hexadecane. Concentrations of berberine below its MIC caused an eightfold increase in release of lipoteichoic acid from the streptococci. Higher concentrations of berberine directly interfered with the adherence of streptococci to host cells either by preventing the complexing of lipoteichoic acid with fibronectin or by dissolution of such complexes once they were formed. Thus, berberine sulfate interferes with the adherence of group A streptococci by two distinct mechanisms: one by releasing the adhesin lipoteichoic acid from the streptococcal cell surface and another by directly preventing or dissolving lipoteichoic acid-fibronectin complexes.

Alkanes↗