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Streptolysin O (SLO) and streptolysin S (SLS) are potent cytolytic toxins produced by almost all clinical isolates of group A streptococci (GAS). Allele-replacement mutagenesis was used to construct nonpolar (in-frame) deletion mutations in the slo and sagB genes of the serotype M5 GAS strain Manfredo, producing isogenic single and double SLO- and SLS-defective mutants. In contrast to recent reports on SLS-defective insertion mutants (I. Biswas, P. Germon, K. McDade, and J. Scott, Infect. Immun. 69:7029-7038, 2001; Z. Li, D. Sledjeski, B. Kreikemeyer, A.Podbielski, and M. Boyle, J. Bacteriol. 181:6019-6027, 1999), none of the mutants described here had notable pleiotropic effects on the expression of other virulence factors examined. Comparison of isogenic parent and mutant strains in various virulence models revealed no differences in their abilities to multiply in human blood or in their 50% lethal doses (LD(50)s) upon intraperitoneal infection of BALB/c mice. A single log unit difference in the LD(50)s of the parent and SLS-defective mutant strains was observed upon infection by the subcutaneous (s.c.) route. Comparisons over a range of infective doses showed that both SLO and SLS contributed to the early stages of infection and to the induction of necrotic lesions in the murine s.c. model. Individually, each toxin made an incremental contribution to virulence that was not apparent at higher infective doses, although the absence of both toxins reduced virulence over the entire dose range examined. Interestingly, in some cases, the contribution of SLO to virulence was clear only from an analysis of the double-mutant strain, highlighting the value of not confining virulence studies to mutant strains defective in the expression of only single virulence factors.

An isogenic mutant of Streptococcus pyogenes Manfredo that lacks the ability to make streptococcal acid glycoprotein (SAGP) has been constructed by inserting a deletion in the sagp gene using the method of allelic exchange. An assay of cell extracts (CE) prepared from the wild-type and mutant Manfredo strains for the enzyme arginine deiminase (AD) showed that significant activity was present in wild-type CE but none could be detected in mutant CE. These findings confirm our earlier conclusion that SAGP has AD activity (B. A. Degnan, J. M. Palmer, T. Robson, C. E. D. Jones, M. Fischer, M. Glanville, G. D. Mellor, A. G. Diamond, M. A. Kehoe, and J. A. Goodacre, Infect. Immun. 66:3050-3058, 1998). Wild-type CE but not mutant CE potently inhibited human peripheral blood mononuclear cell proliferation in response to phytohemagglutinin, and this inhibition was overcome by the addition of L-arginine to proliferation assay mixtures. Invasion assays showed that the isogenic mutant organisms lacking SAGP, and thus AD activity, were between three and five times less able to enter epithelial cells (Hep-2C and A549) than were the wild-type streptococci. Both wild-type and mutant S. pyogenes bacteria were extremely sensitive to low pH. However, L-arginine (1 mM or above) significantly increased the viability of the wild type but not the isogenic mutant organisms under acidic conditions. The difference in acid susceptibility between wild-type and mutant bacteria may explain the reduced capacity of the isogenic mutant bacteria to invade and survive intracellularly.

Streptococcus pyogenes (group A Streptococcus) cell extracts (CE) have a remarkably powerful and dose-dependent inhibitory effect on antigen, superantigen, or mitogen-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro. Purification of the inhibitory component present in S. pyogenes type M5 (Manfredo strain) CE by anion-exchange chromatography followed by gel filtration chromatography showed that the inhibitor had an approximate native molecular mass of 100 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitory fractions followed by silver staining gave a single band with an approximate molecular mass of 47 kDa, indicating that the inhibitor is composed of two identical subunits. NH2-terminal sequencing of the protein revealed that it was identical to the previously characterized streptococcal acid glycoprotein (SAGP); this protein possesses between 31.5 and 39.0% amino acid identity with arginine deiminase (AD) from Mycoplasma hominis, Mycoplasma arginini, Pseudomonas putida, and Pseudomonas aeruginosa. AD enzyme activity was present in unfractionated CE prepared from a range of streptococcal strains, and partially purified inhibitory fractions of Manfredo CE also had high levels of activity. The inhibitory effect of Manfredo CE was overcome by the addition of L-arginine to proliferation assays in which human PBMC were stimulated with phytohemagglutinin. We conclude that SAGP, or its homolog, possesses AD activity and that the potent inhibition of proliferation of human T cells by streptococcal CE is due to activity of this enzyme.

To resolve apparent discrepancies in the literature, N-terminal sequences of the active high- and low-molecular-weight (high- and low-M(r)) forms of native streptolysin O (nSLO) purified from Streptococcus pyogenes culture supernatants and of the similar-size high- and low-M(r) forms of recombinant SLO (rSLO) found in the periplasm of Escherichia coli expressing a cloned slo gene were determined. The high-M(r) forms of nSLO and rSLO are identical, reflecting removal of a 31-residue signal peptide, but the similar-size low-M(r) forms are very different. Removal of C-terminal sequences by proteases in the E. coli periplasm produces an inactive low-M(r) form of rSLO. In contrast, an active low-M(r) form of nSLO is produced by proteolytic cleavage between the N-terminal residues Lys-77 and Leu-78, which was shown to correspond to an extremely sensitive cleavage site for the pyrogenic exotoxin B-derived streptococcal cysteine protease.

We have previously defined major histocompatibility complex (MHC) class II-restricted T-cell epitopes from the carboxy-terminal region of group A streptococcal type 5 M protein. In this report, T-cell responses to one of these epitopes have been characterized in detail. T-cell clones from recombinant M5-immunized mice and popliteal lymph node cells from peptide-immunized mice were used to show that sM5[300-319] is recognized in the context of I-A alleles of four of nine independent MHC class II haplotypes: I-Ad, I-Af, I-Ak, and I-As. This epitope was also recognized by both helper (Th2) and inflammatory (Th1) subsets of murine T cells. The I-Ad-restricted epitope recognized by BALB/c mice was mapped to the 12-amino-acid peptide sM5[308-319] and was shown to provide helper function for an immunoglobulin G anti-peptide antibody response in BALB/c mice. Anti-peptide antibody was shown to be specific for M5[304-315] but failed to recognize intact rM5, suggesting that the conformation of the epitope differed between peptide and protein. However, the results demonstrate that overlapping epitopes can be the focus for both immunoglobulin G antibodies and the T cells which augment their production. Comparison of the available sequences for M proteins indicated that the T-cell epitope within M5[300-319] was highly conserved between M types and hence may elicit helper function for protective antibody responses to a wide range of M types. T-cell epitopes from conserved regions of M proteins which are recognized in the context of multiple MHC haplotypes have potential for the design of multivalent streptococcal vaccines.

Group A streptococcal cell surface M proteins elicit highly protective, serotype-specific opsonic antibodies and many serotypes also elicit host cross-reactive antibodies, which may contribute to the pathogenesis of poststreptococcal autoimmune disease. To date, studies aimed at designing safe (non-host-cross-reactive, defined-epitope) M vaccines have focused almost exclusively on antibody epitopes. Here we identify T-cell epitopes recognized by T cells from BALB/c, C57BL/6, and CBA/Ca mice immunized with purified, recombinant serotype 5 M protein (rM5). The responses of rM5-specific, major histocompatibility complex class II-restricted, T-cell clones to synthetic peptides representing most of the M5 sequence identified at least 13 distinct T-cell recognition sites, including sites recognized by more than one major histocompatibility complex haplotype of mice. Although none of these sites appeared to be strongly immunodominant, an N-terminal peptide, sM5[1-35], was recognized by lymph node T cells of rM5-immunized mice and by a larger proportion of rM5-specific T-cell clones than any other individual peptide. The fine specificity of these clones was mapped with subpeptides to a single site at or overlapping the sequence ELENHDL at residues 21 to 27, which is in close proximity to previously mapped protective antibody epitopes. Other T-cell recognition sites are distributed throughout the M protein and include several in the highly conserved C-terminal region of the molecule. One of these C-terminal sites, located within residues 300 to 319, was recognized by a significant proportion of T-cell clones from two strains of mice. Helper T-cell epitopes located in the C-terminal region of M5 are likely to be widely conserved between different M serotypes and could be particularly useful in designing multivalent, defined-epitope M vaccines.

The abilities of antigen-presenting cells (APC) from nine independent major histocompatibility complex haplotypes and a number of intra-H-2 recombinant congenic strains of mice to present staphylococcal enterotoxin B (SEB) and induce proliferation in murine T-cell receptor V beta 8+ T-cell clones were compared. SEB presented by APC of all haplotypes tested induced significant responses in each of the T-cell clones. The magnitude of response was similar for most haplotypes, but there were limited quantitative differences between certain haplotypes. SEB presented by APC from H-2b mice as well as the intra-H-2 recombinant strains B10.GD and B10.A(4R), which do not express cell surface I-E (designated I-E-), induced the poorest T-cell responses. However, APC from AfE-, AsE-, and AqE- mice were as potent in SEB presentation as APC expressing both I-A and I-E. Antibodies against I-E were more effective than anti-I-A antibodies at inhibiting responses to SEB presented by APC expressing both I-A and I-E, whereas responses induced by APC expressing I-A but not I-E were blocked by antibodies against I-A. Thus, our results show that I-A can present SEB efficiently but that expression of both I-A and I-E on the same APC results in presentation of SEB predominantly by I-E. In addition, experiments using four distinct I-E- strains of mice indicate that I-A alleles differ in their ability to present SEB.

The complete nucleotide sequence of a cloned streptolysin O (SLO) gene and the amino acid sequence of SLO, predicted from the DNA sequence, are reported. SLO contains a single cysteine residue located close to the C terminus of the molecule and shares extensive structural homologies with other thiol-activated toxins, which allow us to predict functionally important features.

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.

The biological properties of Streptococcus pyogenes M protein cloned and expressed in S. sanguis were investigated. The spm-5 gene previously cloned into Escherichia coli was subcloned into the E. coli-S. sanguis shuttle plasmid pVA838 to produce a newly constructed plasmid, pBK100. Cells of S. sanguis transformed with pBK100 expressed 53-, 55-, and 58-kilodalton polypeptides reacting with type 5 M protein antiserum in immunoblots. The M protein was expressed on the surface of S. sanguis cells as shown by the capacity of the intact cells to (i) inhibit the reactivity of anti-type 5 antibodies with purified M protein as demonstrated by enzyme-linked immunosorbent assay; (ii) inhibit the opsonization by M5 antisera of type 5 S. pyogenes; (iii) express M-protein-like fibrils on the surface of the organisms that react with M5 antisera as revealed by immunoelectron microscopy; (iv) bind plasma fibrinogen and, as a consequence, resist phagocytosis by human blood neutrophils; and (v) be rendered susceptible to phagocytosis by opsonic M5 antisera. These results provide additional evidence that streptococcal M proteins bind host proteins as a ploy to evade host defense mechanisms.

A gene bank of group A Streptococcus strain Manfredo (M protein serotype 5) was constructed with a bacteriophage lambda vector-Escherichia coli K-12 host system and screened by immunoblotting hybrid phage plaques with antisera raised to purified pep M5 (serotype 5 M protein fragment released from the streptococcal cell surface by pepsin). Hybrid phage expressing M5 antigen (lambda M5) were detected in the gene bank at an unexpectedly high frequency. The cloned streptococcal DNA sequences from one lambda M5 phage were subcloned into an E. coli plasmid vector. The M5 gene (smp5) was mapped, and its transcriptional orientation was determined by isolating and characterizing deletion and transposon insertion mutants of the M5+ hybrid plasmid pMK207. This analysis indicated that the intact smp5 gene had been cloned. Anti-pep M5 sera reacted with five pMK207-encoded polypeptides having relative molecular sizes of 64,000, 56,000, 55,500, 52,500, and 50,000. All of these polypeptides were encoded by the same DNA sequences, and all reacted with antisera raised to a synthetic peptide corresponding to the amino-terminal end of pep M5, suggesting that proteolytic cleavage at the carboxy-terminal end of the smp5 gene product generates at least some of the lower relative molecular size forms. Southern blotting experiments with smp5 gene sequences as probes identified multiple copies of DNA sequences sharing homology with the smp5 gene in the type 5 group A streptococcal genome.

The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.