Lipoteichoic acid and M protein: dual adhesins of group A streptococci.

The roles of lipoteichoic acid (LTA) and M protein in the adherence of group A streptococci to human cells were investigated. Both M+ and M- streptococci bound to pharyngeal and buccal epithelial cells in similar numbers. Streptococcal attachment was inhibited by LTA, but not by the pepsin-extracted, amino-terminal half of M protein (pep M), suggesting that M protein does not mediate attachment to these cells. However, a purified, recombinant, intact M protein did block attachment of streptococci to buccal cells. Using synthetic peptides, the inhibitory domain was localized to a region of intact M protein that is within or near the bacterial cell wall. Evidence is presented to suggest that on the surface of streptococci this region of the M protein is probably not accessible for interactions with host cell receptors and that M protein does not mediate attachment to buccal or pharyngeal cells. In contrast, approximately 10-times more M+ streptococci bound to Hep-2 cells than did M- streptococci and pep M protein blocked binding of streptococci to Hep-2 cells. The data suggest that at least two streptococcal adhesins, LTA and M protein, are involved in the adherence of streptococci to certain cells and that the relative contributions of these adhesins to the attachment process depends on the type of host cells used to study adherence.

Metabolically active antigen presenting cells are required for human T cell proliferation in response to the superantigen streptococcal M protein.

M protein from type 5 group A streptococci has been identified as a member of the family of polyclonal T cell activators termed superantigens because it preferentially stimulates T cells bearing specific V beta elements of the T cell receptor (TCR). In this study the molecular and cellular requirements for presentation of this protein to T cells were investigated. Only accessory cells (AC) expressing class II major histocompatibility complex (MHC) molecules were capable of supporting T cell activation in response to a 22 kDa fragment of M protein (pep M). Despite the need for class II elements, processing of pep M5 by the antigen-presenting cells (APC) was not required for T cell proliferation induced by pep M5. Fixation of APC by paraformaldehyde (PF) treatment impaired their ability to induce optimal T cell proliferation in response to pep M5 without significantly affecting interleukin (IL-2) production. In contrast, PF-fixation of cells from the B cell lymphoma line, Raji, did not affect their ability to present pep M5 to human T cells. Addition of rIL-1 and IL-6 to PF-treated APC restored pep M5-induced blastogenesis. Our data suggest that pep M5 directly associates with HLA class II molecules forming a complex that can induce IL-2 production but not optimal proliferation by T cells. Additional signals provided by the AC are required to trigger optimal T cell proliferation in response to this superantigen.

Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.

A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.

Genetic analysis of 987P adhesion and fimbriation of Escherichia coli: the fas genes link both phenotypes.

The 987P fimbrial gene cluster has recently been shown to contain eight genes (fasA to fasH) clustered on large plasmids of enterotoxigenic Escherichia coli and adjacent to a Tn1681-like transposon encoding the heat-stable enterotoxin STIa. Different genetic approaches were used to study the relationship between 987P fimbriation and adhesion. TnphoA mutagenesis, complementation assays, and T7 RNA polymerase-promoted gene expression indicated that all of the fas genes were involved in fimbrial expression and adhesion. In contrast to other fimbrial systems, the lack of expression of any single fas gene never resulted in the dissociation of fimbriation and adhesion, indicating that the adhesin is required for fimbrial expression and suggesting that FasA, the fimbrial structural subunit itself, is the adhesin. In addition, fimbrial length was shown to be modulated by the levels of expression of different fas genes.

987P fimbrial gene identification and protein characterization by T7 RNA polymerase-induced transcription and TnphoA mutagenesis.

The 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987. Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase. The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation. In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined. Several of the gene products demonstrated typical properties of exported proteins. Precursor and processed forms could be correlated after inhibiting protein transport with ethanol. The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results. One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681-like transposon.

Identification of an epitope of type 1 streptococcal M protein that is shared with a 43-kDa protein of human myocardium and renal glomeruli.

The localization of opsonic and tissue-cross-reactive epitopes within the amino terminus of type 1 streptococcal M protein was investigated by using murine mAb raised against synthetic peptides of type 1 M protein. Two mAb (IIIA2 and IIIB8) reacted with epitopes located within amino acid residues 1-12 of type 1 M protein. These antibodies opsonized type 1 streptococci and did not cross-react with human kidney and heart tissue. Another mAb (IC7) reacted with mesangial cells of renal glomeruli and human myocardium. The cross-reactive epitope of mAb IC7 was localized to position 13-19, indicating that it is not the same epitope as the previously described vimentin-cross-reactive epitope at position 23-26 of type 1 M protein. In Western blots of mesangial cell and myocardial proteins, mAb IC7 cross-reacted with a 43-kDa protein. Neither vimentin nor actin inhibited the binding of mAb IC7 to the cross-reactive protein, as determined by Western blot or immunofluorescence inhibition tests. These results provide evidence that type 1 M protein contains at least one autoimmune epitope shared with both human glomeruli and myocardium.

Identification of functional epitopes of Pseudomonas aeruginosa exotoxin A using synthetic peptides and subclone products.

The structure-function relationship of P. aeruginosa exotoxin A (ETA) was examined using synthetic peptides and genetically engineered ETA deletion mutants. Antibodies directed against synthetic peptides have allowed the identification of three ETA epitopes, two within domain I and one within the last 33 amino acids of domain III. In addition two distinct neutralizing determinants have been identified by antibodies directed against subclone products. One was associated with the amino-terminal half of ETA, the proposed receptor binding region. The second was associated with the carboxy-terminal half of ETA, a region previously not associated with receptor-binding. The amino-terminal subclone also offers potential as an ETA vaccine, since it produces a stable, non-enzymatically active product, effective in inducing ETA neutralizing antibodies. Data derived from these studies were used in a re-evaluation of structure-function relationships between ETA and diphtheria toxin.

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen.

Stimulation of T cells by superantigens has been reported to be dependent on the presence of APC where binding to class II molecules is a prerequisite to recognition by the TCR. We examined the response of human T cells and a leukemic T cell line, Jurkat to the superantigen, streptococcal M protein. We show that immobilized or cross-linked streptococcal M protein stimulates Jurkat cells (V beta 8), but not normal purified human T cells, to produce IL-2. Activation of purified T cells by this superantigen required costimulatory signals provided by PMA, IL-1, and IL-6. These cytokines and growth factors alone can induce IL-2 production by T cells; however, proliferation occurred only in the presence of superantigen, which together with PMA, IL-1, and IL-6 induced the expression of IL-2R alpha on T cells. Similar results were obtained when the response of purified T cells to another known superantigen, staphylococcal enterotoxin B were examined, indicating that this phenomenon is not unique to M protein. Superantigens interact with a large number of T cells with particular V beta, and thus provide excellent models for studies of the role of biochemical events and signal transduction in T cell activation. Understanding these events may also explain the pathogenesis of autoimmune diseases associated with certain superantigens, such as streptococcal M protein that is thought to be involved in rheumatic fever and rheumatic heart disease.

Superantigenicity of streptococcal M protein.

M proteins that define the serotypes of group A streptococci are powerful blastogens for human T lymphocytes. The mechanism by which they activate T cells was investigated and compared with the conventional T cell mitogen phytohemagglutinin, and the known superantigen staphylococcal enterotoxin B. Although major histocompatibility complex (MHC) class II molecules are required for presentation, there is no MHC restriction, since allogeneic class II molecules presented the bacterial protein to human T cells. Type 5 M protein appears to bind class II molecules on the antigen-presenting cells and stimulate T cells bearing V beta 8 sequences. Our results indicate that this streptococcal M protein is a superantigen and suggest a possible mechanism of its role in the pathogenesis of the postinfectious autoimmune sequelae.

Stimulation of oxidative burst in human monocytes by lipoteichoic acids.

Lipoteichoic acid isolated from Streptococcus faecalis or Streptococcus pyogenes caused direct activation of the respiratory burst in human peripheral blood monocytes. This activity appears to be related to the ability of lipoteichoic acid to bind to the monocyte membrane and trigger the polarization of receptors (capping).

Differential signal requirements in T-cell activation by mitogen and superantigen.

The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca2+ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca2+ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.

The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded.

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.

Human and murine antibodies cross-reactive with streptococcal M protein and myosin recognize the sequence GLN-LYS-SER-LYS-GLN in M protein.

Molecular mimicry or epitope similarity between group A streptococcal M proteins and myosin may contribute to the presence of heart reactive antibodies in acute rheumatic fever. In our study overlapping synthetic peptides copying the entire sequence of PepM5 protein were used to map the myosin cross-reactive epitopes of streptococcal M protein recognized by mouse and human mAb and affinity purified myosin-specific antibodies from acute rheumatic fever and rheumatic heart disease sera. Overlapping M protein peptides SM5(164-197)C and SM5(184-197)C inhibited the murine mAb reactions with PepM5 protein. The human mAb and affinity purified myosin-specific antibodies reacted exclusively with SM5(184-197)C. However, one of the five different purified myosin-specific antibodies not only reacted with SM5(184-197)C but also reacted with SM5(84-116)C. The synthetic subpeptides SM5(175-184)C and SM5(188-197C) did not react with any of the antibodies to PepM5 and myosin demonstrating a requirement of the 184-188 amino acid sequence for antibody recognition. A heptapeptide containing the sequence SM5(183-189) was also found to inhibit selected human myosin-specific antibodies and a human antimyosin mAb. Therefore, the majority of mouse and human myosin crossreactive antibodies recognized an epitope within the 14 residue carboxy terminus of PepM5 which appeared to involve the GLN-LYS-SER-LYS-GLN sequence.

Ultrastructural localization of the fibrinogen-binding domain of streptococcal M protein.

Binding of fibrinogen to the M protein located on the surface fibrillae of group A streptococci impedes deposition of complement and thus contributes to the virulence of these organisms. We investigated this binding by electron microscopy using postembedding immunogold labeling. Both fibrinogen and its D fragment formed a distinct dense layer in the surface fibrillae, separated by 10 nm from the compact part of the cell wall. Labeling the sections with anti-fibrinogen or anti-fragment D showed that the fibrinogen-binding region lay within a 25-nm segment of the fibrillae beginning approximately 30 nm from the inner surface of the cell wall. The outer surface of the fibrinogen layer could be labeled with antibody to the amino-terminal half of type 24 M protein, indicating that the fibrillar tips remained exposed after fibrinogen binding. The degree of labeling with anti-fibrinogen, determined by gold particle counting, was the same whether the bacterial cells had been incubated with purified fibrinogen or whole plasma. These results indicate that the fibrinogen-binding region lies in the distal (amino-terminal) half of the M protein molecule but excludes the most distal portion, which is the site of epitopes that interact with opsonic anti-M antibody, and that plasma proteins other than fibrinogen, a number of which are known to bind to group A streptococci, do not interfere with fibrinogen binding.

Vimentin-cross-reactive epitope of type 12 streptococcal M protein.

The NH2-terminal amino acid sequence of type 12 M protein was determined by automated Edman degradation of a 38-kilodalton polypeptide fragment purified from a limited pepsin digest of intact type 12 streptococci. The sequence of the first 13 amino acid residues of the polypeptide confirmed that predicted by the nucleotide sequence of the mature type 12 M protein. A chemically synthesized peptide copying the NH2-terminal 25 residues, SM12(1-25)C, evoked opsonic antibodies against type 12 streptococci as well as renal glomerular cross-reactive antibodies. The serum from one of six rabbits reacted in immunofluorescence tests with human glomeruli in a mesangial staining pattern. The cross-reactive antibodies were completely inhibited by the immunizing peptide and absorption with type 12 streptococci. Subpeptides of the 25-residue synthetic peptide were without inhibitory effect, suggesting that the cross-reactive antibodies are directed against a conformational epitope of SM12(1-25)C. Anti-SM12(1-25)C antisera reacted specifically with the intermediate filament protein vimentin extracted from mesangial cells. None of the cross-reactions of anti-SM12(1-25)C were inhibited by a synthetic peptide SM1(1-26)C of type 1 M protein, which was previously shown to share a cross-reactive epitope with vimentin. These results indicate that type 12 M protein contains at least one vimentin cross-reactive epitope that is clearly distinct from the tetrapeptide epitope shared with vimentin by type 1 M protein.

Cellular and biochemical responses of human T lymphocytes stimulated with streptococcal M proteins.

Purified group A streptococcal M proteins, pep M5 and pep M6, bearing heart cross-reactive epitopes were compared with pep M24, which lacks such epitopes, in their ability to induce functional differentiation of human T lymphocytes. Lymphocytes activated by pep M5 and pep M6 demonstrated cytotoxic activity against cultured heart cells, whereas pep M24-activated cells differentiated into suppressor T cells, which specifically blocked cytotoxic T lymphocytes against cultured human myocardial cells and not NK cell activity against K562 cells. Pep M5 and not pep M24 induced an increase in the number of CD4, 4B4, helper/inducer T cells. In addition, these M proteins appear to induce different biochemical changes in T lymphocytes. Both pep M5 and pep M24 induced the phosphorylation of a 35-kDa cytoplasmic protein; however, only pep M5 induced the phosphorylation of a 28-kDa membrane protein, primarily in CD4 T cells. These data indicate that the virulent M protein Ag of group A streptococci may exert their effect on the human immune system via different mechanisms. Determining these mechanisms and the biochemical pathways involved in T cell differentiation triggered by these Ag may be important in understanding the pathogenesis of post-streptococcal diseases.

Serine and tyrosine protein kinase activities in Streptococcus pyogenes. Phosphorylation of native and synthetic peptides of streptococcal M proteins.

Two forms of protein kinase activity were isolated from crude extracts of Streptococcus pyogenes and partially purified by ion exchange chromatography and affinity chromatography. The phosphorylation activities were shown to be insensitive to cAMP, required the presence of divalent cations, and eluted from a Sephadex G-200 column with approximate molecular masses of 60 and 45 kDa, respectively. Both enzymes were capable of phosphorylating eukaryotic proteins and synthetic polypeptides in addition to endogenous and heterologous prokaryotic proteins at serine and tyrosine residues. Firm evidence for tyrosine kinase activity was obtained by the use of a tyrosine kinase-specific substrate, a 4:1 glutamate:tyrosine copolymer. Both protein kinases phosphorylated HPr, a phosphocarrier protein of the phosphotransferase system isolated from S. pyogenes and Bacillus stearothermophilus, but failed to phosphorylate HPr isolated from Escherichia coli. Both also phosphorylated a native polypeptide fragment (pep M24) as well as synthetic peptide copies of M protein, the major virulence determinant of group A streptococci. These results indicate that prokaryotic protein kinases are capable of phosphorylating eukaryotic proteins and suggest that the protein kinases of streptococci may play an important role not only in the phosphotransferase system but also in the virulence properties of these organisms.

Serine and tyrosine phosphorylation of 28- and 35-kDa proteins of human T lymphocytes stimulated by streptococcal M protein.

Purified polypeptide fragments of certain surface M proteins of group A streptococci stimulate blastogenesis and the differentiation of cytotoxic T lymphocytes of normal human lymphocytes. The biochemical basis of lymphocyte stimulation by a type M5 protein polypeptide fragment (pep M5) was investigated. Optimal blastogenic doses of pep M5 or phytohemagglutinin stimulated the phosphorylation of several cellular proteins. However, pep M5 but not phytohemagglutinin induced the phosphorylation of 28- and 35-kDa proteins. The 28-kDa protein was shown to be phosphorylated only at serine residues, whereas the 35-kDa protein was phosphorylated only at tyrosine residues. Stimulation of peripheral blood lymphocytes with pep M5 caused a two-fold increase in the CD8+ and CD4+ 4B4+ subpopulations of T lymphocytes. The phosphorylation of the 28-kDa protein appeared to be confined to the CD4+ T cell subpopulation.

Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein, vimentin.

The crossreactivity of antibodies against a renal autoimmune epitope of Streptococcus pyogenes M protein with glomerular mesangial cells was investigated. The antibodies directed against the amino acid sequence Ile-Arg-Leu-Arg of the nephritogenic type 1 M protein reacted in a fibrillar pattern with mesangial cells cultured from isolated glomeruli. In Western blots of urea-extracted mesangial proteins, the antibodies reacted with a 56-kD protein. Monoclonal and polyclonal antibodies identified the 56-kD mesangial protein as vimentin. Two synthetic peptides of human vimentin containing the sequence Arg-Leu-Arg reacted with the autoimmune antibodies raised against a streptococcal M protein peptide. These results provide evidence that the intermediate filament protein vimentin shares autoimmune epitopes with streptococcal M protein.