Isolation and characterization of the cell-associated region of group A streptococcal M6 protein

Pancholi, Vijay; Fischetti, Vincent A.

doi:10.1128/jb.170.6.2618-2624.1988

Cited by 119 publications

(116 citation statements)

References 32 publications

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“…Extensive amino acid sequence similarity between FcRA76 and M proteins in their signal peptides was observed. The deduced amino acid sequence also indicated that FcRA76 contains a proline-rich region and a portion of a C-terminal membrane anchor sequence, both similar to that of M6 and M24 proteins (10). From these results, we postulated that streptococci retain a superfamily of genes that encode surface proteins with heterologous functions: a family of immunoglobulin receptors and a family of antiphagocytic M proteins.…”

mentioning

confidence: 80%

Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

Heath

Cleary

1989

Proc. Natl. Acad. Sci. U.S.A.

136

106

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The partial nucleotide sequence for an Fcreceptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. (7). We previously reported the cloning and expression of an Fc-receptor gene from group A streptococci (8, 9). Our initial results, based on Southern hybridization data, indicated that DNA sequence similarity existed between the Fc-receptor genetic locus (fcrA76) and the M12 gene (emm12) (8). Here we report a partial nucleotide sequence offcrA76 and compare it to several M-protein genes.* Our analysis indicated that the cloned receptor protein (FcRA76) is highly similar to M protein in its secondary structure and in organization of functional domains. Extensive amino acid sequence similarity between FcRA76 and M proteins in their signal peptides was observed. The deduced amino acid sequence also indicated that FcRA76 contains a proline-rich region and a portion of a C-terminal membrane anchor sequence, both similar to that of M6 and M24 proteins (10). From these results, we postulated that streptococci retain a superfamily of genes that encode surface proteins with heterologous functions: a family of immunoglobulin receptors and a family of antiphagocytic M proteins. We proposed that homologous recombination between FcRA76 and M-protein genes is a potential means for streptococci to generate antigenic diversity in their surface proteins. § MATERIALS AND METHODSBacterial Strains, Plasmids, Bacteriophage, and Media. Escherichia coli strains JM83, JM105, and JM109, the plasmid expression vectors pUC9 and pUC19, and phage M13mpl8 and M13mp19 were gifts of J. Messing and have been described (11). E. coli JM83, JM109, and DH5a were grown in LB broth or, after transformation with chimeric pUC plasmids, in LB broth/agar containing ampicillin at 50 jig/ml. E. coli JM105 was grown in M9 medium as described (12). The recombinant plasmid pDH56 and the group A streptococcal strain CS110 have been described (8).Preparation of Streptococcal DNA and Isolation of Plasmid DNA. Preparation of DNA from group A streptococci and of plasmid DNA from E. coli have been described (8).Recombinant DNA Techniques. Restriction endonucleases were purchased from Bethesda Research Laboratories and New England Biolabs, and digestions were performed as recommended by the suppliers. Gel electrophoresis was performed in either 0.7% or 1% agarose gels. The method for transfer of DNA to nitrocellulose has been described (13). Southern (14) and colony (15) blot hybridizations were done using heparin as a blocking agent (16), and standard conditions were used for DNA ligation and radiolabeling and for transformation of E. coli (13).DNA Sequencing and Analysis. Previous results indicated that fcrA76 ...

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mentioning

confidence: 80%

Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

Heath

Cleary

1989

Proc. Natl. Acad. Sci. U.S.A.

136

106

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“…The two Fn-binding proteins of S. dysgalactiae have characteristics typical of cell-wall-associated proteins of Gram-positive cocci (Guss et al, 1990;Hollingshead et al, 1986;Pancholi and Fischetti, 1988). Sequences characteristic of signal peptides are present and both proteins have similar hydrophobic membrane spanning domains and COOH-terminal basic residues.…”

Section: Discussionmentioning

confidence: 99%

“…The upstream region is a unique sequence of approximately 50 amino acids called X,, (starting at residue 763) of which 16 amino acids are prolines. Downstream of the R, region, the proline residues are again more frequent than in the rest of the protein, which is typical for the cellwall-spanning domain (W,) of cell-surface proteins in Grampositive cocci (Pancholi and Fischetti, 1988).…”

Section: Nucleotide Sequences Of Fnba and Fnbb And The Deduced Amino mentioning

confidence: 99%

Two different genes coding for fibronectin‐binding proteins from Streptococcus dysgalactiae

Lindgren

McGavin

Signäs

et al. 1993

European Journal of Biochemistry

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The binding of Streptococcus dysgalactiae to fibronectin involves fibronetcin-binding protein(s) present on the bacterial surface. Previously, we reported the cloning of two different genes coding for cell-wall-associated fibronectin-binding proteins from S. dysgalactiae strain S2 [Lindgren, P.-E., Speziale, P., McGavin, M. J., Monstein, H.-J., Hook, M., Visai, L., Kostiainen, T., Bozzini, S. & Lindberg, M. (1992) J. Biol. Chem. 267, 1924-19311. The two genes, fibA and fnbB, have now been sequenced and the primary amino acid sequences of the two fibronectin-binding proteins, FnBA and FnBB, have been deduced. The two proteins have predicted molecular masses of 117 kDa and 122 kDa, respectively, and are organized in a similar way. The fibronectin-binding activities are localized in repeated motifs, 32-37 amino acids long, in the COOH-terminal regions of the proteins. The two fibronectin-binding proteins have heterologous amino acid sequences, except for the COOH-terminal ends which include the fibronectin-binding repeats. The fibronectin-binding regions of the genes have been fused to IgG-binding domains of protein A, utilizing the IgG-binding capacity of the resulting fusion proteins, to facilitate isolation of the fibronectin-binding domains.Fibronectin (Fn) is a large dimeric glycoprotein (molecular mass 430-470 kDa) which is found in plasma and other body fluids in a soluble form, and is incorporated as microfibrils into the extracellular matrix of most tissues as an insoluble form. The main function of Fn is presumably related to its ability to mediate the substrate adhesion of eucaryotic cells, which involves the binding of specific cell-surface receptors of the integrin family to certain domains in the Fn molecule. The first characterized binding site in Fn for eucaryotic cells contains the amino acid sequence RGD and is located in the central region of the protein (Pierschbacher and Ruoslahti, 1984). Recently, additional recognition sites have been identified (Komoriya et al., 1991). Fn also interacts with other matrix components ; binding sites for collagen, fibrin and sulfated glycosaminoglycans are located in discrete domains of the protein. The present knowledge of the structure and function of Fn has recently been summarized by Hynes (1990).

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“…This position precludes selection for variant antigens by antibody surveillance. Furthermore, these authors (44) suggest that the structure of this region is important in stabilizing the M protein molecule in the cell wall. Thus, mutations in this region would probably be selected against.…”

Section: Discussionmentioning

confidence: 99%

“…Protease protection studies with the M6 molecule showed this region to be embedded in the cell wall (44). This position precludes selection for variant antigens by antibody surveillance.…”

Section: Discussionmentioning

confidence: 99%

Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49

1989

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The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.The M protein, a major virulence factor of Streptococcus pyogenes (group A streptococci), enhances the pathogenicity of the organism by blocking phagocytosis in the nonimmunized host (26,27). Antibodies to M protein develop in infected individuals and confer immunity (34). Streptococcal M protein, however, is antigenically variant, and opsonic antibodies to M protein are largely type specific. Thus, with 70 or more M protein serotypes (35), group A streptococci can successfully avoid immune surveillance.Molecular biological techniques have increased our knowledge of the structure of M protein and its antigenic variability. Five M protein genes have been cloned, and all have been at least partially sequenced (16,24,40,42,46). The M proteins encoded by these sequenced genes have a common framework which includes a conserved signal peptide, a hypervariable amino terminus, and a highly conserved C-terminus. Each gene also appears to contain some internal sequence repetitions, although the extent and degree of similarity of these repeats vary among the genes. Furthermore, regions preceding and following these M protein genes are conserved (16,42). This suggests that the genes are situated in a common expression site on the streptococcal chromosome.Our understanding of how the M protein hypervariable regions evolved is still incomplete. Duplication and deletion of intragenic repeats present in the variable regions of M molecules is a possible mechanism for the introduction of variant amino acids into the M protein sequences (25). Recent evidence shows that intragenic recombination can indeed introduce variant opsonic determinants into the repetitive regions of the M6 protein (28). Not all of the opsonic antigenic determinants of M proteins, however, are located in the repetitive regions of the molecules (25, 32). Thus, additional mechanisms must be involved in the generation of M protein antigenic diversity.A model explaining the generation of M protein antigenic diversity must account for the fact that other streptococcal proteins exhibit antigenic diversity in a nonrandom manner relative to the M type. Among these are the T antigen, a * Corresponding author. (18) and by genetic means (8). Clearly, the processe...

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Isolation and characterization of the cell-associated region of group A streptococcal M6 protein

Cited by 119 publications

References 32 publications

Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

Two different genes coding for fibronectin‐binding proteins from Streptococcus dysgalactiae

Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49

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