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.     

Anti-Hsp65 antibodies recognize M proteins of group A streptococci.

Group A streptococcal M protein and the mycobacterial heat shock protein, hsp65, are strong bacterial immunogens that have been linked to arthritis and autoimmunity. Recent evidence has shown that streptococcal arthritis and adjuvant arthritis may be related to epitopes shared between group A streptococci and hsp65. We investigated the possibility that immunological similarities were shared between streptococcal M protein and hsp65. Antibodies against the 65-kDa heat shock protein of Mycobacterium tuberculosis were tested for reactivity with group A streptococci and purified recombinant M proteins (rM5 and rM6). Rabbit polyclonal anti-hsp65 serum was highly reactive with M type 5 Streptococcus pyogenes and rM5 and rM6 proteins in an enzyme-linked immunosorbent assay (ELISA). A mouse anti-hsp65 monoclonal antibody (MAb), IIC8, reacted with streptococcal M types 5, 6, 19, 24, and 49 in an ELISA but showed no reactivity with an isogenic streptococcal mutant which did not express M protein. Anti-hsp65 MAb IIC8 recognized rM5 and rM6 proteins in the ELISA, and MAbs IIC8 and IIH9 reacted strongly with rM6 protein in Western immunoblots. The binding of M protein by anti-hsp65 MAbs was shown to be inhibited by both hsp65 and M protein. These data show that anti-hsp65 antibodies recognize streptococcal M proteins.     

M proteins of group G streptococci isolated from bacteremic human infections.

We studied seven strains of group G streptococci isolated from clinically severe bacteremic infections in six intravenous drug abusers. These group G strains multiplied luxuriantly in fresh human blood. On electron microscopy, they exhibited surface fibrillae similar to those observed in M-protein-rich group A streptococci, but they were not serologically M typable with a battery of 39 M antisera. Rabbit antisera raised against two of the group G strains (1618 and 1750) opsonized the homologous but not the heterologous isolates and exhibited type-specific Ouchterlony immunoprecipitin reactions. Moreover, antisera raised against peptic extracts of strain 1750 also promoted phagocytic killing of that strain. Anti-1750 reacted in high titer in an enzyme-linked immunosorbent assay against peptic extracts of the homologous strain; these antibodies were removed by absorption with 1750 cells but not by absorption with heterologous strains. These studies represent, to our knowledge, the first analysis of virulence factors of group G streptococci isolated from invasive human disease. The seven epidemiologically related blood isolates of group G streptococci possess distinct type-specific, antiphagocytic surface virulence factors analogous to the M proteins of group A streptococci.     

Immunoglobulin-binding FcrA and Enn proteins and M proteins of group A streptococci evolved independently from a common ancestral protein

Significant sequence homology between M proteins and immunoglobulin (Ig)-binding proteins of group A streptococci suggests that these proteins arose by gene duplication followed by the development of functional diversity due to mutations and intragenic recombinations. The deduced sequence of multiple Ig-binding proteins and M proteins were compared to distinguish between two evolutionary models. Did these functionally distinct genes originate in the distant past from duplication of a common ancestral gene and then functionally evolve independently or did they evolve more recently, one from the other by duplication of a fixed gene? Multiple alignments of conserved sequences of these proteins are consistent with the former hypothesis. Comparison of N termini of Ig-binding proteins revealed less diversity than that of the M proteins' N termini, suggesting that these proteins are under less selective pressure to change.     

A highly conserved region present in transcripts encoding heterologous M proteins of group A streptococci

In group A streptococcal strains of 10 different serotypes, the sequence previously identified as homologous to the structural gene for type 6 M protein (emm6) was found to be transcribed. The conserved sequence, which shows greater than 95% homology among heterologous M proteins, was identified as the 3' third of the gene.     

Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.     

Independence of the nephritogenicity of group A streptococci from their M types

Fluorescein labelled IgG fractions of serum from patients with acute post-streptococcal glomerulonephritis (AGN) stained the glomerular basement membrane of renal tissue from the same and other patients with AGN obtained during the early phase of the disease. Using a spectrofluorometric method it was quantitatively shown that the staining capacity of these serum fractions was reduced after they had been preabsorbed with disrupted nephritogenic group A streptococci (type 12, 49 and a non-typeable strain) or their isolated plasma membranes. No such effect was observed when group A streptococci of types 3, 4, 6, 12, 14 and 25 or their plasma membranes obtained from patients without AGN were used for preabsorption. Plasma membranes of the nephritogenic type 12, 49 and the nontypeable strain diminished the staining capacity of the patients' labelled IgG fractions significantly, regardless of the serotype of the original nephritogenic streptococci isolated from the individual patient. These results indicate the presence of specific nephritogenic, type independent antigens in the plasma membrane of certain group A streptococci which appear to be shared by different serotypes.     

Localization of immunoglobulin A-binding sites within M or M-like proteins of group A streptococci.

Many strains of group A streptococci are capable of binding human immunoglobulin A (IgA) by a nonimmune mechanism. M or M-like proteins constitute a family of structurally diverse molecules which form surface fibrillae, and some of the M or M-like protein forms are responsible for the IgA-binding activity. In this report, the binding site for IgA is localized within two structurally distinct M or M-like proteins, ML2.2 and Arp4. Apart from those structural domains which are common to all M and M-like proteins, ML2.2 and Arp4 lack significant levels of amino acid sequence homology, with the exception of a short segment (ALXGENXDLR) located at residues 21 to 30 of the mature ML2.2 protein. Recombinant fusion polypeptides containing portions of the ML2.2 and Arp4 proteins were expressed in Escherichia coli and tested for binding of human myeloma IgA. A 58-residue polypeptide containing residues 14 to 71 of ML2.2 bound human IgA. The IgA-binding site of Arp4 could be localized to a 53-residue polypeptide containing residues 43 to 95, which encompasses the ALXGENXDLR consensus sequence of Arp4 positioned at residues 50 to 59. Site-specific mutagenesis at three codons within the ALXGENXDLR coding sequence of both the ML2.2 and Arp4 recombinant polypeptides leads to a loss in IgA-binding activity. Thus, the ALXGENXDLR consensus sequence is essential for the nonimmune binding of IgA by both ML2.2 and Arp4. However, the failure to bind IgA by polypeptides which partially overlap the 58- and 53-residue IgA-binding polypeptides of ML2.2 and Arp4, yet contain the ALXGENXDLR consensus sequence, strongly suggests that flanking regions are also critical for IgA binding. In summary, the results indicate that common functional domains bearing significant sequence homology are distributed within regions of M or M-like molecules that are otherwise highly divergent.     

Biofilm formation in clinical isolates of group B streptococci from north India

Many bacterial species are capable of living as biofilm, thought to be the predominant growth mode for bacteria in natural environments. Increasing evidence implicates biofilm as the cause of various human infections. In this study, biofilm formation was demonstrated in group B streptococci (GBS) isolated from different sources in the north Indian community at various pH ranges as well as sugar concentrations and correlated it with different serotypes and surface gene (c alpha) profiles. The capability to form biofilm was better demonstrated in strains from asymptomatic carriers (pregnant women) compared to symptomatic patients. Quantitatively bacterial adherence with host cells was greater in isolates that produced biofilm under neutral conditions. This study assessed the biofilm formation in clinical isolates of GBS, which is a step towards understanding its role in pathogenesis.     

Assay of type-specific M antigens on whole group A streptococci

A novel radioimmunoassay of type-specific M antigens on whole group A streptococcal cells is described. Absorbed rabbit anti-M antisera directed against M types 12 and 49 were used for determining M antigens on intact bacterial organisms. Staphylococcal protein A labelled with¹²⁵I was used as an anti-antibody reagent. The absorbed antisera were tested against ten homologous and 48 heterologous serotypes. All homologous serotypes gave an unequivocal reaction distinct from the weaker reaction with the heterologous serotypes. The type-specificity of the reaction was confirmed by the removal of type-specific antibodies after absorption to purified M protein coupled to Sepharose 4B. The results indicate that the described method is a simple and reliable technique for the recognition of M types of group A streptococci and offers a valuable tool for studies of M antigen in situ.     

Genetic separation of serum opacity factor from M protein of group A streptococci.

Mutagenized cultures of three strains of group A streptococci which were M positive and produced the extracellular serum opacity factor (OF) were screened for mutants which failed to exhibit OF activity on serum agar plates. All cell fractions from three mutants studied in detail, including culture supernatants, protoplast membranes, and cytoplasmic fractions, were completely devoid of activity. Two OF- isolates also lacked OF antigen, whereas the third continued to produce cross-reacting antigen. All three mutants were resistant to phagocytosis and yielded acid-extractable M antigen, indicating that the M protein of each strain was unaltered by the mutations. The separation of the OF+ and M+ phenotypes by mutation establishes that genes which code for the M protein and OF are distinct; therefore, the activities and antigenic determinants of each must be associated with separate, distinct protein molecules.     

Protective monoclonal antibodies recognize heat-labile epitopes on surface proteins of spotted fever group rickettsiae.

Thirty-eight monoclonal antibodies that have not been reported previously were developed from mice immunized with Rickettsia rickettsii, R. conorii, and R. sibirica. Western immunoblotting showed that these monoclonal antibodies are directed against heat-sensitive epitopes which are located on two major surface polypeptides with molecular sizes ranging from 115 to 150 kilodaltons. The detection of the two bands did not depend on the presence of 2-mercaptoethanol. Both bands were destroyed by treatment with proteinase K. Monoclonal antibodies examined by immunofluorescence assay reacted with epitopes that are species specific, group reactive, or shared among a smaller subset of species of spotted fever group rickettsiae. Nine of the monoclonal antibodies were evaluated for their ability to neutralize rickettsial infection and thus protect animals against disease caused by homologous species of rickettsiae. Treatment of rickettsiae with monoclonal antibodies F3-12, F3-14, and F3-36 completely protected guinea pigs against illness caused by the homologous organism R. rickettsii. Monoclonal antibodies F9-5G11 and F15-5B12, derived from mice immunized with R. sibirica, conferred partial protection by delaying the onset and shortening the duration of fever in guinea pigs inoculated with R. sibirica. Monoclonal antibodies F2-15, F2-31, F2-53, and F3-12 protected mice from a lethal infection with R. conorii. Heat-labile epitopes of spotted fever group rickettsial surface proteins are important candidate antigens for development of vaccines to confer protective immunity.     

Role of M protein in adherence of group A streptococci.

The role of the M protein in adherence of group A streptococci to human epithelial cells was directly tested by using an isogenic pair of M+ and M- strains. There was no difference between these strains in the number of streptococcal units that adhered to buccal or tonsillar epithelial cells, indicating the following: (i) that adhesins that are not dependent upon M protein expression are present on the surface of group A streptococci and (ii) that the M protein is not the primary streptococcal adherence ligand. However, the M+ strain adhered to tonsillar epithelial cells as aggregates. This aggregation was dependent on the presence of the M protein, since the isogenic M- strain did not clump. The coaggregation of streptococci suggests that the M protein plays an important role in promoting the formation of microcolonies after initial attachment. Binding to fibronectin, a potential epithelial cell receptor for group A streptococci, was also the same for the isogenic M+ and M- strains as well as for an isogenic strain with a regulatory mutation that decreases the expression of M protein. In summary, the M protein is not the primary streptococcal adhesin, nor is it required to orient the streptococcal adhesin and/or fibronectin receptor.     

Survey of emm-like gene sequences from pharyngeal isolates of group C and group G streptococci collected in Spain

We conducted a nationwide surveillance of the variable 5' emm-like (M-like protein gene) sequences from 214 pharyngeal group C and group G streptococci. Almost 75% of the isolates exhibited emm or emm-like sequences previously described. We identified six new 5' emm-like regions, and almost 23% of the isolates were nontypeable. Five emm-like sequences accounted for more than 50% of the isolates in group C and group G, suggesting horizontal gene transfer between strains of different species.     

Influence of intranasal immunization with synthetic peptides corresponding to conserved epitopes of M protein on mucosal colonization by group A streptococci.

A major virulence factor of group A streptococci is M protein, a surface-exposed fibrillar molecule of which there exist more than 80 distinct serological types. Antigenic variability resides largely in the amino-terminal region of M protein, whereas the carboxy-terminal half of the molecule is highly conserved among different M serotypes. We sought to determine whether mucosal immunization with conserved epitopes of M protein influences the course of mucosal colonization by group A streptococci in a mouse model. Synthetic peptides corresponding to sequences in the conserved region of M protein were covalently linked to the mucosal adjuvant cholera toxin B subunit. Mice were immunized intranasally with the peptide-cholera toxin B subunit conjugate or with cholera toxin B subunit alone and then challenged intranasally with live streptococci. Pharyngeal colonization by streptococci was measured for up to 15 days postchallenge. Mice immunized with synthetic peptides showed a significant reduction in colonization compared with the control group. The data demonstrate that immunity evoked by conserved portions of M protein influences the outcome of group A streptococcal infection at the nasopharyngeal mucosa in a mouse model.     

Prevalence of polyclonal mefA-containing isolates among erythromycin-resistant group A streptococci in Southern Taiwan

A total of 204 nonrepetitive isolates of group A streptococci (GAS), including 107 randomly collected between 1992 and 1995 and 66 and 31 consecutively collected in 1997 and 1998, respectively, from a university hospital in southern Taiwan were examined to determine the prevalence and mechanisms of erythromycin resistance among these isolates. Resistance to erythromycin was detected in 129 isolates (63.2%) by the agar dilution test. Of these, 42 isolates (32.6%) were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and all carried the ermB gene; 4 (3.1%) were assigned to the inducible MLS resistance (iMLS) phenotype, and all harbored the ermTR gene; and 83 (64.3%) were erythromycin resistant but susceptible to clindamycin (M phenotype), and all possessed the mefA gene. Distributed by years, the rates of erythromycin resistance and different phenotypes were 61.7% (53.0% cMLS, 6.1% iMLS, and 40.9% M phenotype) between 1992 and 1995, 62.1% (12.2% cMLS and 87.8% M phenotype) in 1997, and 71. 0% (9.1% cMLS and 90.9% M phenotype) in 1998. Pulsed-field gel electrophoresis showed that all but 2 cMLS isolates were clonal in origin, and 17 clones were detected among the M-phenotype isolates. These results indicate that the high incidence and increasing rate of erythromycin-resistant GAS in southern Taiwan are due to the prevalence of multiple M-phenotype clones and that clindamycin may be the drug of choice for the treatment of infections with GAS in penicillin-hypersensitive patients in this area.     

Functional analysis of IgA antibodies specific for a conserved epitope within the M protein of group A streptococci from Australian Aboriginal endemic communities.

The mucosa is one of the initial sites of group A streptococcal (GAS) infection and salivary IgA (sIgA) is thought to be critical to immunity. However, the target epitopes of sIgA and the function of sIgA in GAS immunity, in particular the role of accessory cells and complement, is largely unknown. We studied the aquisition and the function of sIgA specific for a conserved region epitope, p145 (sequence: LRRDLDASREAKKQVEKALE) of the M protein. Peptide 145-specific sIgA is highly prevalent within an Aboriginal population living in an area endemic for GAS and acquisition of p145-specific sIgA increases with age, consistent with a role for such antibodies in immunity to GAS. Human sIgA and IgG specific for p145 were affinity purified and shown to opsonize M5 GAS in vitro. Opsonization could be specifically inhibited by the addition of free p145 to the antibodies during assay. Opsonization of GAS was totally dependent on the presence of both complement and polymorphonuclear leukocytes, and, moreover, affinity-purified p145-specific sIgA was shown to fix complement in the presence of M5 GAS. These data show that mucosal IgA to this conserved region peptide within the M protein has an important role in human immunity against GAS and may be useful in a broad-based cross-protective anti-streptococcal vaccine.     

Unique and common protective epitopes among different serotypes of group A streptococcal M proteins defined with hybridoma antibodies.

A set of four monoclonal antibodies was produced against a highly purified pepsin extract of type 5 streptococcal M protein. Three of the four antibodies cross-reacted with purified M proteins from heterologous serotypes and opsonized the respective heterologous organisms. Our studies suggest that monoclonal antibodies may be useful in identifying subpeptides of various M proteins containing common, protective epitopes that are capable of evoking antibodies that would protect against several different potentially "rheumatogenic" serotypes.     

Extracellular neuraminidase production by clinical isolates of group B streptococci from infected neonates.

A total of 73 clinical isolates of group B streptococci obtained from diseased infants in 23 states and Puerto Rico were examined for extracellular neuraminidase production. The association of elevated levels of neuraminidase with serotype III isolates was evident in a broad geographical distribution.