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E H Beachey

Publications and source records attributed to E H Beachey.

At least 55 records · Page 3Linked to original sources
PubMed1988

Hyperadhesive mutant of type 1-fimbriated Escherichia coli associated with formation of FimH organelles (fimbriosomes).

The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae. Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa) subunit protein of type 1 fimbriae. Phenotypically, the mutant demonstrated an eightfold-higher mannose-specific hemagglutination titer than the parent strain. On electron microscopy, the mutant strain expressed the same number of fimbriae as the parent strain. However, numerous 10-nm-diameter rounded structures (fimbriosomes) were observed both closely associated with fimbriae and in the culture medium. Fimbriosomes isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner. Dissociation of the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electrophoresis. Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of dissociated type 1 fimbriae and also blocked the adherence of other strains of type 1 fimbriated E. coli to eucaryotic cells. These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant of the D-mannose-sensitive adhesion of type 1 fimbriae.

Bacterial Adhesion↗
PubMed1988

Conservation of protective and nonprotective epitopes in M proteins of group A streptococci.

Carefully controlled hybridization experiments with probes from a cloned serotype 5 M protein (M5) gene (smp5) were performed with DNA isolated from heterologous M types of group A streptococci, and the homologies detected by hybridization were compared with the ability of anti-pepM5 serum to cross-opsonize heterologous M types. As previously reported (J.R. Scott, S.K. Hollingshead, and V.A. Fischetti, Infect. Immun. 52:609-612, 1986), extensive structural homologies exist among the 3' ends of heterologous M protein genes, but there appears to be an increase in sequence variation as one moves towards the 5' ends. However, a clear, predictive correlation between the hybridization patterns and cross-opsonization was not observed. Antibodies raised to a synthetic peptide corresponding to central, conserved sequences adjacent to the C-terminal sides of the pepsin cleavage sites in M5, serotype 6 M protein, and serotype 24 M protein cross-reacted with heterologous acid-extracted M antigens but were not protective and did not bind to intact streptococcal cells, indicating that these epitopes are inaccessible on the intact cell surface. Removal of the N-terminal half of M5, serotype 6 M protein, or serotype 24 M protein by pepsin exposed the conserved epitope on the cell surface. These results suggest that immunoaccessible protective epitopes are confined to the highly variable N-terminal halves of M proteins and that a single, broadly conserved protective M protein epitope does not exist.

Amino Acid Sequence↗
PubMed1988

Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M protein gene and relation to other genes of Streptococcus pyogenes.

The structural gene for the type 24 M protein of group A streptococci has been cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and the 3' and 5' flanking regions was determined. The sequence includes an open reading frame of 1,617 base pairs encoding a pre-M24 protein of 539 amino acids and a predicted Mr of 58,738. The structural gene contains two distinct tandemly reiterated elements. The first repeated element consists of 5.3 units, and the second contains 2.7 units. Each element shows little variation of the basic 35-amino-acid unit. Comparison of the sequence of the M24 protein with the sequence of the M6 protein (S. K. Hollingshead, V. A. Fischetti, and J. R. Scott, J. Biol. Chem. 261:1677-1686, 1986) indicates that these molecules have are conserved except in the regions coding for the antigenic (type specific) determinant and they have three regions of homology within the structural genes: 38 of 42 amino acids within the amino terminal signal sequence, the second repeated element of the M24 protein is found in the M6 molecule at the same position in the protein, and the carboxy terminal 164 amino acids, including a membrane anchor sequence, are conserved in both proteins. In addition, the sequences flanking the two genes are strongly conserved.

Amino Acid Sequence↗
PubMed1988

Bacterial adherence. Adhesin receptor-mediated attachment of pathogenic bacteria to mucosal surfaces.

Pathogenic bacteria adhere to and colonize mucosal surfaces of the susceptible host 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 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. At the epithelial cell surfaces, two classic examples of bacterial adherence are the lipoteichoic acid-mediated attachments of group A streptococcal and the type 1 fimbriae-mediated attachment of Escherichia coli. 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 type 1 fimbriated E. coli, a minor 29-kDa protein located at the tip of the fimbriae interacts with D-mannose residues of glycoprotein receptors on host cells. Similar adhesin-receptor interactions have now been described for a number of pathogenic microbial agents, and undoubtedly play a central role in the early steps of the infectious process.

Bacterial Adhesion↗
PubMed1987

Human cytotoxic T lymphocytes evoked by group A streptococcal M proteins.

Purified group A streptococcal M proteins were used to stimulate peripheral blood lymphocytes from normal adult volunteers. The activated lymphocytes were cytotoxic against cultured human heart cells, as well as liver cells, fibroblasts, and K562 cells, but showed only minimal cytotoxicity against several animal cell types. The cytotoxic activity evoked by type 5 M protein was dose and time dependent. Rabbit antisera against pep M5 that contained heart-crossreactive antibodies partially inhibited cytotoxicity against heart cells, but had no effect on other target cells, suggesting that a fraction of the effector lymphocytes may be recognizing M protein-crossreactive cell surface antigens. All of the cytotoxic activity was recovered from a CD3+ population of lymphocytes obtained from a fluorescence-activated cell sorter, and CD4+ and CD8+ cells were also cytotoxic. M protein-responsive T cell clones were generated that showed specificity for heart and K562 cells, in addition to clones that were cytotoxic against both cell lines. Our data show that streptococcal M protein evokes cytotoxic T lymphocytes against multiple human but not animal target cells. Some of the effector cells may be specific for cultured myocardial cells, but their role in the pathogenesis of rheumatic carditis will require further studies of lymphocytes from patients with acute rheumatic fever and carditis.

Antigens, Bacterial↗
PubMed1987

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein.

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.

Amino Acid Sequence↗
PubMed1987

Protective immunogenicity and T lymphocyte specificity of a trivalent hybrid peptide containing NH2-terminal sequences of types 5, 6, and 24 M proteins synthesized in tandem.

The protective immunogenicity of a hybrid peptide containing tandem copies of types 5, 6, and 24 M protein epitopes was investigated. An NH2-terminal peptide of type 24 M protein was chemically synthesized and then extended to include NH2-terminal peptides of types 6 and 5 M proteins yielding a 34-residue hybrid peptide containing a cysteine residue at its COOH-terminus. When conjugated via the cysteine residue to keyhole limpet hemocyanin (KLH), emulsified in CFA, and injected into rabbits, the synthetic hybrid evoked opsonic antibodies against types 5, 6, and 24 streptococci without stimulating tissue crossreactive immunity. The trivalent hybrid also was capable of priming T lymphocytes in vivo that responded to each of the native serotypes of M protein as well as to the synthetic hybrid peptide in vitro. The primed T cells failed to respond to the individual component peptides contained in the hybrid peptide, suggesting that the hybrid peptide confers conformations resembling the presentations of each of the subpeptides in the respective serotypes of M protein. The brisk immune responses to the type 6 peptide contained in the middle of the tandem hybrid indicates that with judicious placement between proline residues, potentially hidden peptides are readily accessible to the immune system. These results suggest that synthetic tandem peptides can be tailored in a fashion in which each of the component sets of protective epitopes can be made optimally immunoaccessible and immunogenic.

Amino Acid Sequence↗
PubMed1987

Sequence of protective epitopes of streptococcal M proteins shared with cardiac sarcolemmal membranes.

Synthetic peptide fragments spanning the entire amino acid sequence of pep M5 were used to detect epitopes cross-reactive with heart tissue components other than myosin. Heart-cross-reactive pep M5 antibodies were affinity purified by absorption to and elution from purified sarcolemmal membranes. Only one of the synthetic peptides, SM5(164-197)C, inhibited reactivity of the affinity-purified antibodies with pep M5 by ELISA. SM5(164-197)C linked to KLH evoked both opsonic and heart-cross-reactive antibodies in rabbits. In addition to type 5, the immune sera opsonized M types 6, 18, 19, and 49 streptococci. The antisera reacted strongly with isolated cardiac sarcolemmal membranes by immunofluorescence. In Western blots of cardiac tissue, the anti-SM5(164-197)C reacted with a 40 kDa protein but not with myosin. The reaction was inhibited by pep M5 and SM5(164-197)C but not by any of the other peptides spanning pep M5. The cross-reactive anti-SM5(164-197)C affinity purified on sarcolemmal membranes opsonized types 5, 6, and 19 but not type 24 streptococci. These results indicate that SM5(164-197)C contains heart-cross-reactive, opsonic epitopes that are shared among heterologous serotypes of group A streptococci.

Amino Acid Sequence↗
PubMed1987

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein.

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.

Antigens, Bacterial↗
PubMed1987

Hybridoma antibodies to the lipid-binding site(s) in the amino-terminal region of fibronectin inhibits binding of streptococcal lipoteichoic acid.

In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain.

Animals↗
PubMed1987

Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein.

Two murine monoclonal antibodies (immunoglobulin M) evoked against human kidney tissue were examined for cross-reactivity with group A streptococci. A glomerulus-specific antibody, PMII.40.H.2, cross-reacted in an enzyme-linked immunosorbent assay with purified pepsin-extracted M proteins from type 6 and 12 streptococci, but not type 1, 3, 5, 19, and 24 M proteins. The cross-reactive monoclonal antibody also opsonized type 6 and 12 streptococci, indicating that it was directed against protective M protein epitopes that were exposed on the surfaces of these organisms. A control antibody, which was tubule specific, did not cross-react with any of the purified M proteins, nor did it opsonize any of the serotypes of streptococci tested. Western immunoblot experiments identified the glomerular cross-reactive antigen as a 43-kilodalton protein. The results demonstrate that M protein of group A streptococci and glomerular antigens of the human kidney possess cross-reactive determinants.

Animals↗
PubMed1987

Use of monoclonal antibodies to probe subunit- and polymer-specific epitopes of 987P fimbriae of Escherichia coli.

The relationship between the structure and biological function of 987P fimbriae of a strain of enterotoxigenic Escherichia coli (O9:K103:H-) from piglets was investigated. A set of four monoclonal antibodies was prepared from the spleen cells of mice immunized with isolated 987P fimbriae. Antibodies E11, D5, and C3, but not G10, reacted in enzyme-linked immunosorbent assays with 987P fimbriae-bearing E. coli. Electron microscopy showed that E11 and D5 reacted in a discrete periodic pattern forming a spiral motif along the length of the fimbriae. The results of enzyme-linked immunosorbent assays were in agreement with these results; antibodies E11 and D5 reacted at a high dilution (1:12,000) with native fimbriae on the surface of E. coli, whereas antibody C3 reacted at an intermediate dilution (1:3,000) and G10 failed to react at all (less than 1:250). In contrast, C3 and G10 reacted at a dilution of 1:3,276,000 with the fimbrial subunits derived by treating the isolated fimbriae with 6 M guanidine hydrochloride, whereas E11 and D5 reacted with the subunits at much lower dilutions of 1:800 and 1:6,400, respectively. Moreover, fimbriae reassembled from the subunits regained reactivity with antibodies D5 and E11, indicating that these antibodies are directed against quaternary conformational epitopes. Only the three antibodies (D5, E11, and C3) that recognized epitopes accessible on intact fimbriae were able to efficiently block the adhesion of 987P fimbriated E. coli to piglet enterocytes. These results indicate that certain epitopes of 987P fimbriae are dependent on quaternary structural conformation, whereas others are present on monomeric subunits; some of the latter appear to remain accessible on fully assembled fimbriae.

Animals↗
PubMed1987

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.

Antibodies, Bacterial↗
PubMed1987

Assembly of a chemically synthesized peptide of Escherichia coli type 1 fimbriae into fimbria-like antigenic structures.

Escherichia coli type 1 fimbriae are composed of subunits, each of which comprises 158 amino acids. We synthesized a copy of a 13-residue peptide, located near the NH2 terminus of the fimbrial subunit, that assumed some of the properties of type 1 fimbriae. At pH 5.5 the synthetic peptide autoassembled into fibrillar structures that resembled type 1 fimbriae except that they appeared less rigid and rodlike. A quaternary structure-specific monoclonal antibody against type 1 fimbriae recognized the synthetic peptide in the assembled but not the unassembled state. Furthermore, when the synthetic peptide was injected in its fimbrial conformation into rabbits, it evoked antibodies that reacted with type 1 fimbriae isolated from E. coli.

Antibodies, Monoclonal↗
PubMed1986

Sequence of myosin-crossreactive epitopes of streptococcal M protein.

Group A streptococcal M proteins contain epitopes that crossreact with sarcolemmal membrane proteins of human myocardium and myosin. In the present study, synthetic peptide copies spanning the entire 197-residue pepsin extracted fragment of type 5 M protein were used to localize the myosin-crossreactive epitopes of the molecule. Peptide 84-116 inhibited by 75% the binding of myosin-crossreactive antibodies evoked by pep M5, as determined by ELISA. Immunoblot inhibition studies confirmed that peptide 84-116 almost totally inhibited the binding of pep M5 antibodies to the heavy chain of human cardiac myosin. None of the remaining synthetic peptides, including peptide 1-35, which contains protective epitopes, inhibited antibodies binding to myosin. Two of three rabbits immunized with peptide 84-116 developed low but significant levels of antibodies crossreactive with myosin. Identification of the primary structures containing tissue-crossreactive as opposed to protective epitopes should not only allow the development of safe and effective M protein vaccines, but may also provide insights into the pathogenesis of rheumatic heart disease.

Amino Acid Sequence↗