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

DNA sequence analysis of the complete M6 protein gene revealed 19 hydrophobic amino acids at the C terminus which could act as a membrane anchor and an adjacent proline- and glycine-rich region likely to be located in the cell wall. To define this region within the cell wall and its role in attaching the molecule to the cell, we isolated the cell-associated fragment of the M protein. Assuming that the cell-associated region of the M protein would be embedded within the wall and thus protected from trypsin digestion, cells were digested with this enzyme, and the wall-associated M protein fragment was released by phage lysin digestion of the peptidoglycan. With antibody probes prepared to synthetic peptides of C-terminal sequences, a cell wall-associated M protein fragment (molecular weight, 16,000) was identified and purified. Amino acid sequence analysis placed the N terminus of the 16,000-molecular-weight fragment at residue 298 within the M sequence. Amino acid composition of this peptide was consistent with a C-terminal sequence lacking the membrane anchor. Antibody studies of nitrous acid-extracted whole bacteria suggested that, in addition to the peptidoglycan-associated region, a 65-residue helical segment of the C-terminal domain of the M protein is embedded within the carbohydrate moiety of the cell wall. Since no detectable amino sugars were associated with the wall-associated fragment, the C-terminal region of the M6 molecule is likely to be intercalated within the cross-linked peptidoglycan and not covalently linked to it. Because the C-terminal region of the M molecule is highly homologous to the C-terminal end of protein A from staphylococci and protein G from streptococci, it is likely that the mechanism of attachment of these proteins to the cell wall is conserved.     

Epitopes of group A streptococcal M protein that evoke cross-protective local immune responses.

The present studies were undertaken to identify conserved epitopes of group A streptococcal M proteins that evoke cross-protective mucosal immune responses. Two synthetic peptides copying conserved regions of type 5 M protein, designated SM5(235-264)C and SM5(265-291)C, were covalently linked to carrier molecules and their immunogenicity was tested in laboratory animals. Rabbit antisera against both peptides cross-reacted with multiple serotypes of group A streptococci, indicating that the peptides contained broadly cross-reactive, surface exposed M protein epitopes. Serum antipeptide antibodies adsorbed to the surface of heterologous type 24 streptococci passively protected mice against intranasal challenge infections. Mice that were actively immunized intranasally with each synthetic peptide covalently linked to the B subunit of cholera toxin were protected against colonization and death after intranasal challenge infections with type 24 streptococci in the absence of serum opsonic antibodies. These data confirm and extend previous observations that conserved M protein epitopes evoke cross-protective local immunity and may serve as the basis for broadly cross-protective M protein vaccines.     

Conformational characteristics of the complete sequence of group A streptococcal M6 protein

M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were "core" nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.     

Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins

The 1479-base pair (bp) nucleotide sequence of the serotype 5 M protein gene (smp5) from Streptococcus pyogenes contains three distinct types of tandemly repeated sequences, designated A, B, and C. Repeat A (21 bp x 6, in the 5'-half of smp5), shares no homology with the types 6 or 24 M protein genes (Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1986) J. Biol. Chem. 261, 1677-1686; Mouw, A. R., Beachey, E. H., and Burdett, V. (1988) J. Bacteriol., in press). Repeat B (75 bp x 3.6, in the center of smp5) is also present in the M6, but not in the M24 gene. Repeat C (105 bp x 2.7, just distal to the B repeats) shares homology with repeats in both the M6 and M24 genes. All three genes share extensive homology in their 3'-halves and in 5' sequences encoding the N-terminal signal peptides, but between these two regions there are highly variable sequences that are responsible for antigenic diversity. These relationships suggest that both intergenic and intragenic recombination has occurred during the evolution of distinct M protein serotypes. All three M proteins contain conserved hydrophobic and proline-rich sequences at their C-terminal ends, suggestive of a membrane anchor and a peptidoglycan spanning region.     

Sequence homology of group A streptococcal Pep M5 protein with other coiled-coil proteins

Group A streptococcal Pep M5 protein, an antiphagocytic determinant of the bacteria, is an alpha-helical coiled-coil molecule, and exhibits significant sequence homology with tropomyosin and myosin, but to a lesser degree with other coiled-coil proteins. Moreover, Pep M5 is more homologous to myosin than to tropomyosin, and the homologies are more numerous between the C-terminal domain of the Pep M5 protein and the S2 fragment of myosin. The C-terminal domain of the Pep M5 protein exhibits extensive sequence identity with the C-terminal region of Pep M6 molecule, another M protein serotype. Thus, regions within two M protein serotypes are homologous to the S2 region of the myosin molecule. These observations are consistent with the immunological findings of other investigators and thus may explain some of the previously reported immunological cross-reactions between antigens of the group A streptococcus and mammalian heart tissue.     

Epitopes of group A streptococcal M protein shared with antigens of articular cartilage and synovium

Rabbit antisera evoked by purified pepsin-extracted group A streptococcal M proteins were screened for the presence of joint cross-reactive antibodies by indirect immunofluorescence using thin sections of mouse knee joints. Pep M1, M5, and M18 antisera contained antibodies that cross-reacted with chondrocytes, cartilage, and synovium. Immunofluorescence inhibition assays showed that some of the joint cross-reactive epitopes were shared among the three heterologous serotypes of M protein. The pep M5 joint cross-reactive epitopes were localized to three different synthetic peptides of the C-terminal region of pep M5. Immunoblot analyses showed that the M5 joint cross-reactive antibodies recognized two proteins of human synovium and cartilage of molecular mass 56 and 58 kDa. The cross-reactive antibodies binding to the 56-kDa protein were inhibited by purified vimentin in immunoblot inhibition experiments. M protein-specific antibodies from patients with acute rheumatic fever were also shown to cross-react with joint tissue in a pattern similar to the rabbit antisera. Rabbit and human M protein-specific antibodies that were bound to articular cartilage activated significant levels of complement when compared to control serum, suggesting that M protein joint cross-reactive antibodies could potentially be involved in the pathogenesis of ARF and arthritis.     

M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis

Background  

Group A streptococcal (GAS) infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF) and rheumatic heart disease (RHD). RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT) by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assay for molecular typing the M protein gene (emm) of GAS.     

Chemistry and end-group analysis on purified M protein of type 12 group A streptococcal cell walls

M protein was extracted from the cell walls of streptococci by use of both acidic and alkaline buffers. These extracts were further purified by ammonium sulfate fractionation and column chromatography. Both diethylaminoethyl and carboxymethyl celluloses were employed to cover the pH range of 3.0 to 9.0. All of the M proteins isolated were immunologically related, but their physical and chemical properties varied dependent upon the pH range of isolation. Each isolate appeared to be homogeneous on the basis of immunodiffusion analysis, electrophoretic mobility, and ultracentrifugal analysis, but their amino acid analyses differed slightly. Two factors were shared by all isolates: (i) they all reacted with type-specific antisera and (ii) each seemed to have l-lysine as a single N-terminal amino acid.     

Tropomyosin shares immunologic epitopes with group A streptococcal M proteins

Tropomyosin is an alpha-helical coiled-coil protein with structural similarities to the streptococcal M protein. In order to show serologic cross-reactivity between streptococcal M proteins and tropomyosin, we selected from a panel of murine mAb those which reacted with M proteins and tropomyosins in the ELISA. Western blots were used to study the reactions of each mAb with human and rabbit cardiac and rabbit skeletal tropomyosins. The antibodies were further characterized for their reactions with the additional autoantigens myosin, actin, keratin, and DNA. Five mAb were found which reacted with either PepM5 or ColiM6 protein and tropomyosin in Western blots or ELISA. Two of the tropomyosin positive mAb were also antinuclear antibodies and were inhibited with DNA. In Western blots of cardiac tropomyosins, the mAb reacted with either the 70-kDa dimer of tropomyosin, the 35-kDa monomer, or both. Some differences were observed in the reactions of the mAb with the different tropomyosins in Western blots. The heart cross-reactive epitopes shared between M proteins and tropomyosin were in most instances shared with cardiac myosin. Differences were observed among the reactions of the mAb with the different tropomyosins. This report constitutes the first evidence of serologic cross-reactivity between streptococcal M proteins and tropomyosins.     

Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population

Background  

Most group A streptococcal (GAS) vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains.     

The plasminogen-binding group A streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine residues

The migration of the human pathogen Streptococcus pyogenes (group A streptococcus) from localized to deep tissue sites may result in severe invasive disease, and sequestration of the host zymogen plasminogen appears crucial for virulence. Here, we describe a novel plasminogen-binding M protein, the plasminogen-binding group A streptococcal M protein (PAM)-related protein (Prp). Prp is phylogenetically distinct from previously described plasminogen-binding M proteins of group A, C, and G streptococci. While competition experiments indicate that Prp binds plasminogen with a lower affinity than PAM (50% effective concentration = 0.34 microM), Prp nonetheless binds plasminogen with high affinity and at physiologically relevant concentrations of plasminogen (K(d) = 7.8 nM). Site-directed mutagenesis of the putative plasminogen binding site indicates that unlike the majority of plasminogen receptors, Prp does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys(96) and Lys(101) reduced but did not abrogate plasminogen binding by Prp. Plasminogen binding was abolished only with the additional mutagenesis of Arg(107) and His(108) to alanine. Furthermore, mutagenesis of Arg(107) and His(108) abolished plasminogen binding by Prp despite the presence of Lys(96) and Lys(101) in the binding site. Thus, binding to plasminogen via arginine and histidine residues appears to be a conserved mechanism among plasminogen-binding M proteins.     

Size variation in group A streptococcal M protein is generated by homologous recombination between intragenic repeats

Summary M protein, a major surface protein and virulence factor for the group A streptococcus, exhibits extraordinary size variation in strains of the same serotype (Fischetti et al. 1985). RNA sequence analysis of spontaneous M protein size variants shows that deletion mutations arise in a single strain by homologous recombination events between intragenic tandem repeats. Similar deletion and duplication events also occur in serial streptococcal isolates from a single patient and among related strains in a recent outbreak. We discuss how homologous recombination events can lead to the generation of antigenic variation.     

The amino-terminal region of group A streptococcal M protein determines its molecular state of assembly and function

Group A streptococcal M protein, a major virulence factor, is an alpha-helical coiled-coil dimer on the surface of the bacteria. Limited proteolysis of type 57 streptococcus with pepsin released two fragments of the M57 molecule, with apparent molecular weights of 32,000 and 27,000 on SDS-PAGE. However, on gel filtration under nondenaturing conditions, each of these proteins eluted as two distinct molecular forms. The two forms corresponded to their dimeric and monomeric state as compared to the gel filtration characteristics of known dimeric coiled-coil proteins. The results of sedimentation equilibrium measurements were consistent with this, but further indicated that the dimeric form consisted of a dimer in rapid equilibrium with its monomer, whereas the monomeric form does not dimerize. The monomeric form was the predominant species for the 27 kD species, whereas the dimeric form predominated for the 32 kD species. Sequence analysis revealed the 27 kD species to be a truncated derivative of the 32 kD PepM57 species, lacking the N-terminal nonheptad region of the M57 molecule. These data strongly suggested that the N-terminal nonheptad region of PepM57 is important in determining the molecular state of the molecule. Consistent with this, PepM49, another nephritis-associated serotype, which lacks the nonheptad N-terminal region, also eluted as a monomer on gel filtration under nondenaturing conditions. Furthermore, removal of the N-terminal nonheptad segment of the dimeric PepM6 protein converted it into a monomeric form. The dimeric molecular form of both the 32 kD PepM57 and the 27 kD PepM57 did not represent a stable state of assembly, and were susceptible to conversion to the corresponding monomeric molecular forms by simple treatments, such as lyophilization. The 27 kD PepM57 exhibited a greater propensity than the 32 kD species to exist in the monomeric form. The 32 kD species contained the opsonic epitope of the M57 molecule, whereas the 27 kD species lacked the same. This is consistent with the previous reports on the importance of the N-terminal region of M protein for its opsonic activity. Together, these results strongly suggest that, in addition to its importance for the biological function, the N-terminal region of the M protein plays a dominant role in determining the molecular state of the M molecule, as well as its stability.     

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.     

Nucleotide substitutions and small-scale insertion produce size and antigenic variation in group A streptococcal M1 protein

The presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type-specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N-terminal non-repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N-terminal non-repeat domains of two other closely related strains, 71-155 and 76-088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N terminal non-repeat domain of the M protein of GAS.     

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.     

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

Characteristics of group A streptococcal bacteriophages

Friend, Patric L. (Northwestern University Medical School, Chicago, Ill.), and Hutton D. Slade. Characteristics of group A streptococcal bacteriophages. J. Bacteriol. 92:148-154. 1966.-A medium for the growth of group A streptococcal phages is described, consisting of Brain Heart Infusion broth supplemented with 0.2% yeast extract, 10(-4)m CaCl(2), and 10 mug/ml of dl-tryptophan. Cell and phage growth in this medium was excellent, and did not require the addition of serum or other proteins as indicated by other workers. Growth of one phage has also been achieved in a completely synthetic medium. The adsorption characteristics of two group A phages in protein broth and synthetic broth were studied, and the initial adsorption of phage was found to be more extensive in synthetic broth. However, the final amounts of adsorption in both were similar. The addition of purified group A carbohydrate antigen to the adsorption mixture in synthetic broth had no effect on the adsorption, and cells containing type-specific M protein adsorbed phage at the same rate as those lacking M protein. It was concluded that neither the group antigen nor the type antigen was the primary site of phage adsorption. One-step growth curves of the two phages showed a second step or burst occurring. Sonic oscillation of the bacterial cultures, which broke up the chains to single cells, abolished the second step of the growth curve. It appears that the second step is a function of the chain formation of streptococcal cells.     

Angiosperm phylogeny inferred from sequences of four mitochondrial genes

An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.