M6 Protein Research Articles

Peripheral blood T cell responses to keratin peptides that share sequences with streptococcal M proteins are largely restricted to skin-homing CD8(+) T cells.

The association of psoriasis with Streptococcus pyogenes throat infections suggests a potential antigenic target for the T cells that are known to infiltrate psoriatic skin. Streptococcal M protein share an extensive sequence homology with the human epidermal keratins. Keratin 17 (K17), while being mostly absent from uninvolved skin, is up-regulated in psoriatic lesions. Consequentially, M-protein-primed T cells may recognize up-regulated keratin epitopes via molecular mimicry. Using in vitro lymphocyte culture and cytokine flow cytometry we demonstrate that HLA-Cw*0602(+) psoriasis patients had significant CD8(+) T cell interferon (IFN)-gamma responses to peptides from the K17 and M6 protein selected on the basis of sequence homology and predicted HLA-Cw*0602 binding. These responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (CLA(+)) subset of CD8(+) T cells. CD4(+) T cells showed only borderline responses. CLA(+) CD8(+) T cells from Cw6(+) non-psoriatic individuals responded to some M6 peptides but rarely to K17 peptides. Cw6(-) psoriasis patients showed a response that was intermediate between Cw6(+) patients and controls. These findings indicate that psoriatic individuals have CD8(+) T cells that recognize keratin self-antigens and that epitopes shared by streptococcal M proteins and human keratins may be targets for the CD8(+) T cells that infiltrate psoriatic skin lesions.

Characterization of a complement-binding protein, DRS, from strains of Streptococcus pyogenes containing the emm12 and emm55 genes.

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.

Peripheral Blood T‐Cell Responses to Keratin Peptides that Share Sequences with M Proteins are Largely Restricted to Skin‐Homing CD8+ T Cells

The association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the T cells that are known to infiltrate dermis and epidermis of psoriatic skin. Streptococcal M protein shares an extensive sequence homology with human epidermal keratins. Keratins 16 (K16) and 17 (K17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. There is increasing evidence that CD8+ T cells play an important effector role in psoriasis and M protein‐primed T cells may recognize these shared epitopes in skin via molecular mimicry. To identify candidate epitopes, peptides with sequences from K17 were selected on the basis of predicted binding to HLA‐Cw6 and sequence similarities with M6 protein. Matched peptides from the sequence of M6 protein and a set of peptides with poor predicted binding were also selected. Cw6+ individuals with psoriasis and Cw6+ healthy controls, having a family history of psoriasis, were recruited. PBMCs were incubated with the peptide antigens. T‐cell activation in the CD4+, CD8+ and later the skin‐homing cutaneous lymphocyte‐associated antigen (CLA)‐expressing subset of CD8+ T cells was evaluated by CD69 expression and intracellular IFN‐γ accumulation using flow cytometry. We demonstrate that Cw6+ psoriasis patients had significant CD8+ T‐cell IFN‐γ responses to peptides from K17 and M6 protein selected on the basis of sequence homology and predicted HLA‐Cw*0602 binding. These responses were about 10 times more frequent in the skin‐homing cutaneous lymphocyte‐associated antigen‐expressing (CLA+) subset of CD8+ T cells. CD4+ T cells showed only borderline responses. CD8+ T cells from Cw6 + nonpsoriatic individuals responded to some M6 peptides but very rarely to K17 peptides, and this also applied to the CLA+CD8+ subset. These findings indicate that psoriatic individuals have CD8+ T cells that recognize keratin self‐antigens and that epitopes shared by streptococcal M protein and human keratin may be targets for the CD8+ T cells that infiltrate psoriatic skin lesions.

CD4 + T-cells from peripheral blood of a patient with psoriasis recognize keratin 14 peptide but not ‘homologous’ streptococcal M-protein epitope

Psoriasis has been recognized as an immunologically mediated inflammatory skin disease that has been associated with group A, β-haemolytic streptococcal infections. Notably cross-reactive autoimmune mechanism, which is mediated by T cells reacting to epitopes that are common to streptococcal M-protein and keratin, has been proposed in psoriasis. In order to investigate this possibility, peptides corresponding to M-protein and human epidermal keratin, which share some amino acid sequence between them, were synthesized and tested for their ability to stimulate T-cells of patients with psoriasis. Among five cases examined, we isolated a CD4 + T-cell line that recognized the type I keratin (K14) p168–181 when it was presented by the patient's HLA-DR molecules from a single psoriatic patient, whose MHC allele was HLA-A2/A26, -B27/B16, -DR4/DR8, -DQ8. Further analysis disclosed that the critical peptide recognized by the T-cell line was 10-mer keratin p171–180 (DLRNKILTAT). However, corresponding M6 protein with homology to K14 did not stimulate the T-cell response and no evidence for cross-reactivity was obtained. The K14-responsive T cell line produced IFN-γ, but little IL-4 when stimulated with irradiated autologous PBMC pulsed with this peptide. Thus, the finding that human epidermal keratin peptide is immunogenic in a psoriasis patient may provide the evidence that T lymphocytes play an important role in the pathogenesis of psoriasis as an autoimmune disorder participated with Th1 like cells. However, the keratin-responsive T cell line was detected in only one of five cases of psoriasis examined, suggesting that such T cell line appears to be not so popular in psoriatic patients. No evidence for cross-reactivity to streptococcal M protein also suggests that the contribution of streptococci may simply be inducing proliferation of various repertoire of T cells (including K14-responsive T cells) possibly through a superantigen-dependent process.

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46).

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.

Analysis of factors affecting surface expression and immunogenicity of recombinant proteins expressed by gram-positive commensal vectors.

Several key protein structural attributes were altered in an effort to optimize expression and immunogenicity of a foreign protein (M protein from Streptococcus pyogenes) exposed on the surface of Streptococcus gordonii commensal bacterial vectors: (i) a shorter N-terminal region, (ii) the addition of a 94-amino-acid spacer, and (iii) the addition of extra C-repeat regions (CRR) from the M6 protein. A decrease in the amount of cell surface M6 was observed upon deletion of 10 or more amino acid residues at the N terminus. On the other hand, reactivity of monoclonal antibody to surface M6 increased with the addition of the spacer adjacent to the proline- and glycine-rich region, and an increase in epitope dosage was obtained by adding another CRR immediately downstream of the original CRR. The results obtained should facilitate the design of improved vaccine candidates using this antigen delivery technology.

Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors

To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-γ) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-γ to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.

Skin T cell proliferative response to M protein and other cell wall and membrane proteins of group A streptococci in chronic plaque psoriasis.

To determine and compare the T cell response to M protein and other group A streptococcal (GAS) antigens, T cell lines (TCL) were cultured from the lesional skin of 33 psoriatic patients and 17 disease controls. GAS-reactive skin TCL were tested in proliferation assays with recombinant M6 protein, and extracts of cell wall and membrane from type M6 GAS and its corresponding M gene deletion mutant. Initially, GAS-reactive skin TCL were obtained from 16 of 25 (64%) psoriasis, and from seven of 17 (41%) control patients. Eleven psoriatic and four control GAS-reactive TCL proliferated to M6 cell wall extract, whereas all the TCL from both groups responded to the extract of M6 membrane proteins. This difference in response to the two extracts was significant for both groups of patients (psoriasis, P = 0.0335, controls, P = 0.0156). GAS-reactive TCL from a further eight psoriasis patients showed no difference in response to cell wall extract from M6 GAS (containing the M protein minus its C-terminus) compared to that of its corresponding M gene deletion mutant. Furthermore, GAS-reactive TCL did not proliferate to recombinant M6 protein. However, a small, but significant reduction in proliferation by the eight psoriatic GAS-reactive TCL to the M-negative (lacking the M protein C-terminus) compared to M6-positive membrane extract was observed (P = 0.01). These findings suggest that GAS-reactive T cells in skin lesions of chronic plaque psoriasis proliferate to streptococcal membrane and, to a lesser extent, cell wall proteins. However, psoriatic skin T cells do not recognize cell wall M protein.

Surface expression of the conserved C repeat region of streptococcal M6 protein within the Pip bacteriophage receptor of Lactococcus lactis.

The C repeat region of the M6 protein (M6c) from Streptococcus pyogenes was expressed within the Pip bacteriophage receptor on the surface of Lactococcus lactis. M6c was also detected in the culture medium. The pip-emm6c allele was integrated into the chromosome and stably expressed without antibiotic selection. The level of cell-associated surface expression of PipM6c was 0.015% of total cellular protein. The amount of PipM6c on the cell surface was increased about 17-fold by expressing pip-emm6c from a high-copy-number plasmid. Replacing the native pip promoter with stronger promoters isolated previously from Lactobacillus acidophilus increased surface expression of PipM6c from the high-copy-number plasmid up to 27-fold. Concomitantly, the amount of PipM6c in the medium increased 113-fold. The amount of PipM6c did not vary greatly between exponential- and stationary-phase cultures. Western blots indicated that the full-length PipM6c protein and most of the numerous proteolytic products were found only on the cell surface, whereas only one proteolytic fragment was found in the culture medium.

Stronger proliferative response to membrane versus cell-wall Streptococcal proteins by peripheral blood T cells in chronic plaque psoriasis.

Proliferative responses of peripheral blood mononuclear cells (PBMC) to group A streptococcal (GAS) antigens have been studied in 24 patients with psoriasis and 15 disease controls. Extracts of cell wall (including M protein) from types M4 and M12 GAS, recombinant M6 protein, and both cell-wall and cell-membrane extracts from type M6 (M6+) GAS and its corresponding M gene deletion mutant (M6-) were tested. PBMC from psoriatic patients proliferated more strongly to cell-wall extracts from M12 versus M4 (P = 0.0348), and to M6+ versus M6- (P = 0.0019) GAS with, in most cases, moderate proliferation to recombinant M6 protein. The psoriatic response to M12 cell wall was significantly increased compared to the controls (P = 0.0032). In psoriatics, M6+ membrane extracts induced a markedly greater proliferation than those of cell wall (P = 0.0002); responses to M6+ (P = 0.0039) and M6- (P = 0.0114) membrane extracts were higher than those of the control PBMC. Both groups showed a decreased response to the M6- versus M6+ membrane extracts (P = 0.0030; P = 0.0181, respectively). This study has demonstrated that patients with psoriasis have a heightened circulating T-cell response to cell wall M protein and to non-M proteins present on the cell wall and membrane of GAS.

Genetic dissection of the Streptococcus pyogenes M1 protein: regions involved in fibronectin binding and intracellular invasion

Entry of serotype M1 Streptococcus pyogenes into host cells depends on binding of the host glycoprotein fibronectin (Fn) by the bacterial M1 protein. The present study was undertaken to localize the Fn binding region in M1 and assess other potential functions of M1. A set of recombinant M1 protein fragments were assayed for their capacities to bind Fn and inhibit ingestion of streptococci by epithelial cells. M1 protein, M6 protein and internally-deleted derivatives of M1 were expressed on the surface of Lactococcus lactis. Lactococci that expressed M1 or M6 protein bound Fn and were efficiently taken up by epithelial cells. Deletion of both the N-terminal A and B repeats regions of M1 abrogated Fn binding and intracellular invasion. Deletion of either the A domain (M1ΔA) or B repeats (M1ΔB) significantly reduced, but did not completely eliminate, Fn binding indicating that M1 protein may possess two independent Fn binding sites. Fn binding by the M1ΔA or M1ΔB proteins was insufficient for efficient invasion, however, suggesting that M protein binding alters the structure of Fn that, in turn, affects the interaction between Fn and epithelial cells. Although expression of M1, M6 or M1ΔB proteins led to aggregation of lactococcal cells, aggregation did not significantly contribute to invasion efficiency.

Generation and surface localization of intact M protein in Streptococcus pyogenes are dependent on sagA.

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019-6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor.

CLEAVAGE AND PURIFICATION OF INTEIN FUSION PROTEINS USING THE STREPTOCOCCUS GORDONII SPEX SYSTEM

A Gram-positive bacterial expression vector using Streptococcus gordonii has been developed for expression and secretion, or surface anchoring of heterologous proteins. This system, termed Surface Protein Expression system or SPEX, has been used to express a variety of surface anchored and secreted proteins. In this study, the Mycobacterium xenopi (Mxe) GyrA intein and chitin binding domain from Bacillus circulans chitinase A1 were used in conjunction with SPEX to express a fusion protein to facilitate secretion, cleavage, and purification. Streptococcus gordonii was transformed to express a secreted fusion protein consisting of a target protein with a C-terminal intein and chitin-binding domain. Two target proteins, the C-repeat region of the Streptococcus pyogenes M6 protein (M6) and the nuclease A (NucA) enzyme of Staphylococcus aureus, were expressed and tested for intein cleavage. The secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cleavage to release the target protein. The M6 and NucA fusion proteins were shown to bind chitin beads and elute under cleavage reaction conditions. In addition, NucA demonstrated enzyme activity both before and after intein cleavage.

Design of a protein-targeting system for lactic acid bacteria.

We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (approximately 95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.

Non-M protein(s) on the cell wall and membrane of group A streptococci induce(s) IFN-gamma production by dermal CD4+ T cells in psoriasis.

Recently we have demonstrated that a disease-specific subpopulation of CD4+ T cells isolated from skin lesions of chronic plaque psoriasis produces interferon-gamma in response to group A streptococcal (GAS) antigens. To determine if these T cells recognize M or non-M protein, extracts from cell wall of type M6 GAS (M6W) and its isogenic M gene deletion mutant (M-W), M6 membrane extract (M6M) and recombinant M6 protein (rM6) were used to stimulate GAS-reactive T-cell lines from nine patients with chronic plaque psoriasis. T-cell lines were incubated with or without streptococcal extracts for 18 h in the presence of a transport inhibitor, stained for surface CD4 and intracellular cytokine expression, and analysed by flow cytometry. Variable numbers (0.2-34%) of CD4+ T cells produced interferon-gamma, in all but one of the T-cell lines tested, in response to M6W, M-W and M6M extracts. No significant difference between the response to M6W and M-W extracts was detected. In addition, rM6 protein failed to increase CD4+/interferon-gamma+ T-cell numbers in seven of nine T-cell lines compared to medium alone. For the group, there was a highly significant correlation between the responses to the three extracts (M6W vs M-W, P = 0.0005; M6W vs M6M, P = 0.0003; M-W vs M6M, P = 0.0001). Low or minimal numbers of interleukin-4- and interleukin-10-producing CD4+ T cells were occasionally induced. These findings suggest that a subpopulation of CD4+ T cells isolated from skin lesions of chronic plaque psoriasis patients produces interferon-gamma in response to non-M protein(s) present on the cell wall and membrane of GAS.

Increased titers of antibodies against streptococcal M12 and M19 proteins in patients with Tourette's syndrome

It has been suggested that a post-streptococcal autoimmune process may be involved in the pathogenesis of a subgroup of children with tics and obsessive–compulsive symptoms (PANDAS). Elevated antibody titers against streptococcal antigens have also been described in adult patients suffering from Tourette's syndrome (TS). In order to characterise further streptococcal antigens, we focussed on M proteins. M proteins are a major virulence factor of group A streptococci and known to evoke an immunologic cross-reaction with diverse epitopes of human tissue including brain tissue. Therefore, antibodies against M proteins may play a role in the pathophysiology of at least a subgroup of TS patients. Antibodies against M proteins were studied in 25 adult patients suffering from TS and 25 healthy controls after careful medical examination. The antibody titers against the peptides M1, M4, M6, M12 and M19 were estimated by ELISA. Our results show increased titers of antibodies against the streptococcal M12 and M19 proteins in TS patients as compared with controls, while antibody titers against M1, M4 and M6 did not differ between the TS and control groups. Elevated serum titers of antibodies against M12 and M19 proteins support the view that a streptococcus-induced autoimmune process may be involved in TS. The finding of a possible autoimmune origin of TS has implications for both pathophysiology and future therapeutic strategies.

Identification of an intragenic integration site for foreign gene expression in recombinant Streptococcus gordonii strains.

A new intragenic chromosomal integration site within the lacG gene of the lac operon has been identified in Streptococcus gordonii for use in the expression of foreign genes. Introduction of a portion of the Streptococcus pyogenes emm6 gene into the lacG locus resulted in the lactose-inducible surface expression of the S. pyogenes M6 protein. This result demonstrates the ability to modulate the in vitro or in vivo expression of a foreign gene in a S. gordonii recombinant using a biosynthetic metabolite.

Expression and purification of histidine-tagged proteins from the gram-positive Streptococcus gordonii SPEX system.

Streptococcus gordonii (S. gordonii) has been used as a gram-positive bacterial expression vector for secreted or surface-anchored recombinant proteins. Fusion of the gram-positive bacterial N-terminal signal sequence to the target protein is all that is required for efficient export. This system is termed SPEX for Surface Protein EXpression and has been used to express proteins for a variety of uses. In this study, the SPEX system has been further developed by the construction of vectors that express polyhistidine-tagged fusion proteins. SPEX vectors were constructed with an N-terminal or C-terminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogenes M6 protein and the Staphylococcus aureus nuclease A (NucA) enzyme were tested for expression. The fusion proteins were purified using metal affinity chromatography (MAC). Results show that the fusion proteins were expressed and secreted from S. gordonii with the His tag at either the N- or C-terminal position and could be purified using MAC. The M6 fusions retained immunoreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The M6-His and NucA-His fusions were purified approximately 15- and 10-fold respectively with approximately 30% recovery of protein using MAC. This study shows that the polyhistidine tag in either the N- or C-terminal position is a viable way to purify secreted heterologous proteins from the supernatant of recombinant S. gordonii cultures. This study further illustrates the value of the SPEX system for secreted expression and purification of proteins.

Engineering the Gram-Positive Cell Surface for Construction of Bacterial Vaccine Vectors

A genetic system for surface display of heterologous proteins has been developed in Streptococcus gordonii, a gram-positive human oral commensal that is naturally competent for genetic transformation. Our approach is based on chromosomal integration downstream from a resident promoter and translational fusion to an M6 protein. Using this strategy a variety of proteins, of different origin and size, were displayed on the cell surface and were shown to be stably expressed both in vitro and in vivo. Animal models of mucosal colonization (oral and vaginal) and intragastric immunization with recombinant S. gordonii were developed and the local and systemic immune responses were studied. Here we report the techniques for the construction of recombinant bacteria, use of animal models, and analysis of the immune response.

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells.

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.