Antigenic epitope mapping of the M24 protein of Streptococcus pyogenes: implications for serodiagnosis of rheumatic fever

Abstract Rheumatic fever continues to be a significant problem in Australian Aboriginal communities and developing countries worldwide. Early diagnosis could facilitate the institution of penicillin prophylaxis resulting in the prevention of recurrences of rheumatic fever. An overlapping biotinylated peptide bank of 82 peptides, based on the known sequence of Streptococcus pyogenes M24 protein, was used in a standard enzyme immunoassay. A total of 82 sera were tested from both aboriginal and non-aboriginal subjects with clinically proven rheumatic fever, rheumatic heart disease and matched controls. Two peptides with significant sequence homology at the C-terminal end were found to be discriminatory between aboriginal cases and controls. It is proposed that these peptides could be the basis of a serological test for rheumatic fever.     

Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation

Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes , which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.     

Type 1 M protein of Streptococcus pyogenes. N-terminal sequence and peptic fragments

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of Mr 20270 as the main product which covers the N-terminal part of the M protein. The amino acid sequence was determined of the N-terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N-terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N-terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.     

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.     

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.     

Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein

Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR) of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP), a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.     

Cellular aspects of the distinct M protein and SfbI anchoring pathways in Streptococcus pyogenes

Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis.     

Synthesis and immunological activity of an oligosaccharide-conjugate as a vaccine candidate against Group A Streptococcus

The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS–TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.     

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.     

Analysis of plasminogen-binding M proteins of Streptococcus pyogenes

Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogen-binding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.     

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

The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.     

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.     

Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.     

Bacteriophage content of M49 strains of Streptococcus pyogenes

Bacteriophages are common autonomous migrating mobile genetic elements in group A Streptococcus (GAS) and are often associated with the carriage of various virulence genes, including toxins, mitogens and enzymes. Two collections of GAS type M49 strains isolated from invasive (22 strains) and noninvasive (16 strains) clinical cases have been studied for the presence of phage and phage-associated virulence genes. All the GAS strains carried from at least two to six phage genomes as determined by the number of known phage integrase genes found. A sampling of the invasive M49 strains showed that they belonged to the same multilocus sequence typing type, carried two specific integrase genes ( int 5 and int 7), and contained the toxin genes spe A, spe H and spe I. Other invasive strains lacking this gene profile carried the prophage integrating in mutL–mutS region and inducing the 'mutator' phenotype. We suggest that this specific phage-related virulence gene constellation might be an important factor increasing M49 GAS pathogenicity.     

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.     

Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria.

The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelope-associated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.     

Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes

The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.