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V A Fischetti

Publications and source records attributed to V A Fischetti.

At least 127 records · Page 7Linked to original sources
PubMed1984

Immunological studies of post-streptococcal sequelae. Evidence for presence of streptococcal antigens in circulating immune complexes.

Since elevated levels of circulating complexes have been noted to occur in the sera of patients with post-streptococcal sequelae, the possibility that these complexes contained streptococcal antigens within the complex was investigated. Sera from these patients were precipitated with polyethylene glycol to extract a fraction rich in these complexes, which was then injected into rabbits. The rabbit sera were then reacted with both cellular and extracellular fractions obtained from streptococcal strains associated with either acute post-streptococcal nephritis (APSGN) or acute rheumatic fever (ARF) by using immunoelectrophoresis and ELISA techniques. The data demonstrate that both ARF and APSGN complexes contain streptococcal antigens. However, APSGN complexes react uniquely to certain extracellular antigens present in those strains associated with nephritis, while ARF complexes react specifically to certain streptococcal extracellular antigens excreted by strains associated with rheumatic fever. Neither of the two groups of complexes appear to contain streptococcal antigens related to any cellular antigens derived from the group A streptococcus. Additionally, a rabbit serum immunized with streptococcal extracellular products reacted directly with complexes isolated from nephritis patients. Removal of the gamma globulin by absorption with an anti-human Fc serum resulted in the concomitant loss of reactivity with the anti-streptococcal serum, strongly suggesting an intimate association of the streptococcal antigen with these complexes. The presence of streptococcal antigens within the circulating immune complex of patients with APSGN coupled with their specific presence in those strains associated with post-streptococcal glomerulonephritis argues strongly for a causal role of these antigens in the disease process.

Antibodies, Bacterial↗
PubMed1983

Expression of streptococcal M protein in Escherichia coli.

The structural gene for group A streptococcal M protein, the fibrillar surface molecule enabling the organism to resist phagocytosis, has been cloned into Escherichia coli. The molecule produced by Escherichia coli is slightly larger than the M protein isolated by solubilization of the streptococcal cell wall, but is similar in size to that secreted by streptococcal protoplast and L forms. Immunologically, the molecule synthesized by Escherichia coli has the same type-specific determinants as the streptococcal M protein.

Antigens, Bacterial↗
PubMed1983

Toxic shock syndrome and lysogeny in Staphylococcus aureus.

Lysogeny, or the presence of temperate bacteriophage, was demonstrated, by means of two Staphylococcus aureus indicator strains, in 11 of 12 strains of S. aureus isolated from patients with toxic shock syndrome. Only 1 of 18 strains of S. aureus that were not associated with toxic shock syndrome showed the presence of bacteriophage. A laboratory strain of S. aureus was lysogenized by bacteriophage from two of the toxic shock-associated strains. These results add support to the theory that lysogeny by one or more bacteriophage in certain strains of S. aureus may be responsible for the pathogenesis of toxic shock syndrome.

Bacterial Toxins↗
PubMed1983

Primary structure of streptococcal Pep M5 protein: Absence of extensive sequence repeats.

Extensive sequence repeats have been observed in a biologically active fragment of type 24 streptococcal M protein, namely Pep M24 [Beachey, E. H., Sayer, J. M. & Kang, A. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3163-3167]. To determine whether such extensive repetition in sequence is a common characteristic of the antiphagocytic streptococcal M proteins, we have determined the sequences of the clostripain peptides of Pep M5, a biologically active fragment of the type 5 M protein that is analogous to Pep M24. These sequences, together with the amino-terminal sequence of the whole molecule, accounted for nearly two thirds of the Pep M5 molecule. However, extensive identical repeats of the kind observed in Pep M24 were not present in Pep M5. Preliminary study of the amino acid sequence analysis of the M protein from type 6 Streptococcus has also indicated the absence of sequence repeats within the regions of this molecule examined so far. These results suggest that extensive sequence repeats may not be a common characteristic of M-protein molecules. On the other hand, the seven-residue periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil structures, appeared to extend over most of the Pep M5 molecule. This feature has been observed previously for the partial sequences of three M protein serotypes. Thus, the important element of the M-protein structure appears to be the seven-residue periodicity necessary for the maintenance of the coiled-coil structure rather than extensive identical amino acid sequence repeats.

Journal Article↗
PubMed1983

Influence of trifluoroacetic acid on retention times of histidine-containing tryptic peptides in reverse phase HPLC.

The tryptic peptides of the aminoethylated alpha- and beta-chain of hemoglobin have been separated on a Partisil-10 ODS-2 column with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The elution profile of the tryptic peptides of the chains obtained with this acetonitrile trifluoroacetate system has been compared with that obtained using phosphoric acid as the ion-pairing reagent. This comparison demonstrated that trifluoroacetate influences the retention times of the histidine-containing tryptic peptides much more than it affects those peptides that do not contain histidine residues. This behavior has been rationalized on the basis of ion-pairing of trifluoroacetate with the histidine residues of the tryptic peptides.

Chromatography, High Pressure Liquid↗
PubMed1983

Requirements for the opsonic activity of human IgG directed to type 6 group A streptococci: net basic charge and intact Fc region.

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.

Binding Sites, Antibody↗
PubMed1983

Immunological studies of post-streptococcal sequelae: serological studies with an extracellular protein associated with nephritogenic streptococci.

Using the Ouchterlony double diffusion and the crossed-immunoelectrophoresis techniques the reactivity to a purified extracellular product of nephritogenic group A streptococci (NASP) was examined with both acute and convalescent sera obtained from patients with documented post-streptococcal glomerulonephritis and patients with documented acute rheumatic fever. The streptococcal antigen utilized in these studies was first purified on SDS gels and then eluted from the gel, resulting in a single protein band on SDS electrophoresis. Double diffusion studies revealed that only nephritis patients reacted to this extracellular product associated with nephritogenic strains, whereas rheumatic fever sera produced no line of precipitation. An assay of serial bleedings from nephritis patients suggested that the antibody reactive to the NASP was in higher titre in the acute phase of the disease and decreased with convalescence. In confirmation of these findings, crossed-immunoelectrophoresis experiments were conducted with a battery of sera from acute nephritic and non-nephritic patients against the NASP antigen. A striking increase was detected in the reactivity of nephritis patients (96%) compared to non-nephritis sera (15-20%). Comparison between acute and convalescent sera using this technique confirmed the finding of decreasing antibody titre with resolution of disease. These findings of a specific humoral response in patients with acute post-streptococcal nephritis to the NASP of nephritogenic strains further implicates an aetiological function to this protein.

Adolescent↗
PubMed1981

Streptococcal M protein: alpha-helical coiled-coil structure and arrangement on the cell surface.

The conformation and molecular dimensions of purified type 6 streptococcal M proteins establish the close structural relationship of these molecules to tropomyosin. Ultracentrifuge studies reveal that the M molecules exist as stable dimers; circular dichroism spectra indicate that the molecules contain about 70% alpha helix; and fiber x-ray diffraction diagrams show the characteristic reflections of the alpha-helical pattern. Electron microscopic images of M protein shadowed with platinum reveal rod-shaped molecules having the same width as tropomyosin. However, the lengths of the M molecules are about 30% shorter than lengths predicted by assuming a completely alpha-helical molecule. These findings indicate that the structure of the M6 protein is primarily alpha-helical coiled coil. Comparison of the lengths of the fibers on the surface of the streptococcus and the isolated M proteins suggests that each fiber on the cell wall consists of a single M-protein molecule approximately 500 A long. The structure determined for these fimbriae is the first alpha-helical coiled-coil conformation to be demonstrated for bacterial surface projections.

Antibodies, Bacterial↗
PubMed1981

Immunochemical analysis of intact M protein secreted from cell wall-less streptococci.

M protein is a major virulence factor of group A streptococci, which provides these organisms with protection against phagocytosis in the absence of specific antibody. To gain insight into the nature of the native M-protein molecule, type 12 M protein was isolated and purified from the extracellular supernatants of a group A streptococcal L form and stabilized protoplasts. The intact purified M protein from both sources had a molecular weight of 58,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is in contrast to the 32,0000-dalton molecule isolated from the parent type 12 organism by using a nonionic detergent. The purified secretory M protein removed opsonic antibodies from type 12 rabbit immune serum, as demonstrated by a bactericidal assay. Therefore, it appears that either previous nondestructive methods of M-protein isolation have not removed intact M protein from cell walls or part of the molecule is fragmented during its association with cell walls.

Animals↗
PubMed1980

Tropomyosin-like seven residue periodicity in three immunologically distinct streptococal M proteins and its implications for the antiphagocytic property of the molecule.

Partial sequences of three immunologically distinct group A streptococcal M proteins (M5, M6, and M24) revealed significant homology with each other, certain amino acid residues being conserved within the three molecules. In addition, a common feature of the sequenced regions of these M proteins was their high alpha-helical potential and the presence of a repeating seven residue periodicity that is characteristic of the double helical coiled-coil molecule, tropomyosin. The existence of a tropomyosin-like seven residue periodicity strongly suggests that regions of these three M proteins may participate in intra- and/or intermolecular coiled-coil interactions. Because of the constraints imposed by such a repeating periodicity, certain conserved residues within the M proteins would occupy spatially equivalent positions in the tertiary structure of these molecules. This common characteristic could play an important role in the common antiphagocytic property of the immunologically diverse M molecules. In addition to similarities in the secondary structure of M proteins and tropomyosin, significant sequence homology has also been observed between certain regions of these molecules with up to 50% identical residues. As a result of the striking structural similarity with tropomyosin, M proteins may play a regulatory role in the contractile mechanisms involved in phagocytosis.

Amino Acid Sequence↗
PubMed1980

Studies on group A streptococcal M-proteins: purification of type 5 M-protein and comparison of its amino terminal sequence with two immunologically unrelated M-protein molecules.

M-protein was isolated from group A, type 5, streptococci by limited proteolysis with pepsin and purified by chromatography on DEAE-Sephadex followed by gel filtration. The protein thus purified (Pep M5) was homogeneous by SDS-polyacrylamide gradient gel electrophoresis (apparent m.w.: 19,000), retained the capacity to remove opsonic antibodies from type 5 antiserum, and was capable of eliciting opsonic antibodies in rabbits. Its amino acid composition was very similar to that reported for M-proteins from other streptococcal types. The sequence of the first 29 amino terminal residues of Pep M5 was determined and compared with the reported amino terminal sequences of two immunologically unrelated M-proteins, namely, Pep M6 and Pep M24. The results revealed that, although the amino terminal sequences fo these three proteins differed from each other, some amino acid residues appeared to be conserved, suggesting a certain degree of structural relatedness among these M molecules. The possibility that this feature forms the molecular basis for the common antiphagocytic behavior of immunologically unrelated M-proteins is discussed.

Amino Acid Sequence↗
PubMed1979

The occurrence of a protein in the extracellular products of streptococci isolated from patients with acute glomerulonephritis.

The present report compares the extracellular proteins of streptococci by sodium dodecyl sulfate polyacrylamide electrophoresis. A marked variation in the streptococcal extracellualr proteins (SEP) of different strains was detected, even in strains of similar serotypes. It was possible, however, to identify a single protein band that occurred predominantly in the SEP of strains isolated from patients with acute poststreptococcal glomerulonephritis (APSGN). This protein was generally not produced by streptococci obtained from patients without this disease. It appears to be immunologically similar in the various serotypes of streptococci isolated from patients with APSGN and can be demonstrated by immunofluorescence techniques to be present in the glomeruli of these patients.

Antigens, Bacterial↗
PubMed1979

Amino acid sequence and physicochemical similarities between streptococcal M protein and mammalian tropomyosin.

The amino-terminal sequences of two peptides of type 24 streptococcal M protein show similarities with that of rabbit skeletal muscle tropomyosin, having up to 40% identical residues and probabilities of occurring by chance as low as P less than 10(-5). In addition, a hexapeptide (Glu-Ala-Glu-Lys-Ala-Ala) that is found five times in the M24 protein was shown to be identical to a sequence in tropomyosin. Similarities are also seen in the amino acid compositions and physicochemical properties of the two proteins. The amino-terminal sequences of peptides from another bacterial surface protein, staphylococcal protein A, are highly correlated with segments of two other myofibrillar proteins, rabbit actin (P less than 10(-7)) and rabbit myosin A1 light chain (P less than 10(-6)). The data presented suggest that a close structural relationship exists between mammalian muscle proteins and the biologically active surface proteins of staphylococci and streptococci. In addition, the correlation between sequences in M protein and tropomyosin represents direct evidence of a structural similarity at a molecular level between a streptococcal protein and a mammalian muscle component and may therefore prove relevant to the pathogenicity of the streptococcus.

Actins↗
PubMed1978

Streptococcal M protein extracted by nonionic detergent. III. Correlation between immunological cross-reactions and structural similarities with implications for antiphagocytosis.

Three immunologically cross-reactive and non-cross-reactive streptococcal M proteins were analyzed by a chromatographic tryptic peptide mapping system. The results indicate that cross-reactions correlate with the extent of structural similarity among the M protein molecules analyzed. The data also reveal that free lysine is released by the action of trypsin from these three M proteins, suggesting a common lys-lys or arg-lys sequence. In addition, only one peptide has been found to be common within all three M types. This limited structural relatedness among the three M proteins examined indicates that sequence variation plays a major role in the immunological specificity of the M antigens. However, despite sequence variation, all M protein molecules have a common antiphagocytic activity. The fact that no common opsonic antibody has yet been found, even against limited M types, argues against this biological activity being solely the result of a common sequence. Based on these data, it is suggested that the antiphagocytic effect of M protein may be due to a conformationally created environment on the surface of the molecule which is selected by both immunological and biological pressure.

Antigens, Bacterial↗
PubMed1977

Streptococcal M protein extracted by nonionic detergent. II. Analysis of the antibody response to the multiple antigenic determinants of the M-protein molecule.

Purified streptococcal M protein extracted by nonionic detergent was used in an RIA and a solid-phase radiocompetitive inhibition assay to determine the nature of the immune response in both human beings and hyperimmunized rabbits to this complex antiphagocytic antigen. Results indicate that a type-specific response to an M antigen with the development of opsonic antibodies is the result of antibodies directed against the majority of the antigenic determinants of the molecule. Cross-reactions between certain M types on the other hand, are represented by antibodies directed against only a small percentage of these antigenic determinants. Results also suggest that avidity may play a role in the action of opsonic antibodies. However, the data indicate that factors besides avidity (i.e. sites bound by the antibodies) also seem essential for opsonization.

Adult↗
PubMed1977

Streptococcal M protein: an antiphagocytic molecule assembled on the cell wall.

After extraction with nonionic detergent, type 6 streptococcal M protein was found to be composed of multiple proteins ranging in molecular weight from 35,000 to 6,000 daltons. The antiphagocytic proteins, however, were found to be limited to three species having molecular weights of 28,000, 31,000, and 35,000 daltons. These molecules which removed opsonic antibodies from immune serum could be separated from those proteins that had only type specificity. Pulse chase experiments supported by chemical and immunological data suggest that the smaller, type-specific molecules are used to assemble the larger, antiphagocytic proteins. Type 6 M protein was radialabeled and used in a binding assay for the measurement of opsonic antibodies in human serum. Good correlation was observed between binding and the presence of opsonic antibodies in both systems. However, certain sera did exhibit binding but lacked type-specific opsonic activity. Results of competitive inhibition experiments demonstrated that the nonopsonic serum was deficient in certain antibodies that were present in opsonics serum and that the anitphagocytic molecules contained the sites necessary to bind these antibodies.

Amino Acids↗
PubMed1976

Streptococcal M protein extracted by nonionic detergent. I. Properties of the antiphagocytic and type-specific molecules.

Group A streptococcal M protein was extracted with nonionic detergent and subjected to a number of physical, chemical, and immunological tests. M protein thus extracted was composed of multiple protein bands, ranging from 35,000 down to 6,000 daltons, all having type-specific precipitating activity. The anti-phagocytic proteins, however, were limited to three molecular species having mol wt of 28,000, 31,000, and 35,000 daltons, and could be separated from those proteins that had only type specificity. Physical studies indicated that these proteins existed as individual asymmetrical molecules which were not aggregated. By radiolabeling M protein on living streptococci, it was determined that these protein bands were found on the streptococcal cell wall in this multiple form. Also, by pulse chase experiments supported by chemical and immunological data, evidence was obtained strongly suggesting that the smaller, type-specific molecules are used to assemble the larger, antiphagocytic proteins.

Amino Acids↗