Streptococcal M-protein Research Articles

Generation and characterization of a plasminogen-binding group A streptococcal M-protein/streptokinase-sensitive mouse line

BackgroundStreptococcus pyogenes (GAS) is a human bacterial pathogen that generates various mild to severe diseases. Worldwide, there are approximately 700 million cases of GAS infections per year. In some strains of GAS, the surface-resident M-protein, plasminogen-binding group A streptococcal M-protein (PAM), binds directly to human host plasminogen (hPg), where it is activated to plasmin through a mechanism involving a Pg/bacterial streptokinase (SK) complex as well as endogenous activators. Binding to Pg and its activation are dictated by selected sequences within the human host Pg protein, making it difficult to generate animal models to study this pathogen. ObjectivesTo develop a murine model for studying GAS infection by minimally modifying mouse Pg to enhance the affinity to bacterial PAM and sensitivity to GAS-derived SK. MethodsWe used a targeting vector that contained a mouse albumin-promoter and mouse/human hybrid plasminogen cDNA targeted to the Rosa26 locus. Characterization of the mouse strain consisted of both gross and histological techniques and determination of the effects of the modified Pg protein through surface plasmon resonance measurements, Pg activation analyses, and mouse survival post-GAS infection. ResultsWe generated a mouse line expressing a chimeric Pg protein consisting of 2 amino acid substitutions in the heavy chain of Pg and a complete replacement of the mouse Pg light chain with the human Pg light chain. ConclusionThis protein demonstrated an enhanced affinity for bacterial PAM and sensitivity to activation by the Pg-SK complex, making the murine host susceptible to the pathogenic effects of GAS.

Binding of the kringle-2 domain of human plasminogen to streptococcal PAM-type M-protein causes dissociation of PAM dimers.

The direct binding of human plasminogen (hPg), via its kringle‐2 domain (K2hPg), to streptococcal M‐protein (PAM), largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2), with one K2hPg binding a‐repeat in its A‐domain, we employed biophysical techniques to analyze the mechanism of the K2hPg/PAMNS88.2 interaction. We show that apo‐PAMNS88.2 is a coiled‐coil homodimer (M.Wt. ~80 kDa) at 4°C–25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature‐dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg/PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg/PAMNS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg) showing that PAM is a functional monomer at 37°C.

Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence

Virulent strains of Streptococcus pyogenes (gram-positive group A Streptococcus pyogenes [GAS]) recruit host single-chain human plasminogen (hPg) to the cell surface—where in the case of Pattern D strains of GAS, hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R561-V562 peptide bond, resulting in the disulfide-linked two-chain protease, human plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2hPg), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine-binding site of K2hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the lysine-binding site of K2hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mouse Pg emphasizes the importance of the Pg latent heavy chain (residues 1–561) in PAM binding and shows that while SK2b binds to both hPg and mouse Pg, the activation properties of streptokinase are strictly attributed to the serine protease domain (residues 562–791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface.

A local α-helix drives structural evolution of streptococcal M-protein affinity for host human plasminogen.

Plasminogen-binding group A streptococcal M-protein (PAM) is a signature surface virulence factor of specific strains of Group A Streptococcus pyogenes (GAS) and is an important tight binding protein for human plasminogen (hPg). After activation of PAM-bound hPg to the protease, plasmin (hPm), GAS cells develop invasive surfaces that are critical for their pathogenicity. PAMs are helical dimers in solution, which are sensitive to temperature changes over a physiological temperature range. We previously categorized PAMs into three classes (I-III) based on the number and nature of short tandem α-helical repeats (a1 and a2) in their NH2-terminal A-domains that dictate interactions with hPg/hPm. Class II PAMs are special cases since they only contain the a2-repeat, while Class I and Class III PAMs encompass complete a1a2-repeats. All dimeric PAMs tightly associate with hPg, regardless of their categories, but monomeric Class II PAMs bind to hPg much weaker than their Class I and Class III monomeric counterparts. Additionally, since the A-domains of Class II PAMs comprise different residues from other PAMs, the issue emerges as to whether Class II PAMs utilize different amino acid side chains for interactions with hPg. Herein, through NMR-refined structural analyses, we elucidate the atomic-level hPg-binding mechanisms adopted by two representative Class II PAMs. Furthermore, we develop an evolutionary model that explains from unique structural perspectives why PAMs develop variable A-domains with regard to hPg-binding affinity.

Anti-streptococcal antibody and T-cell interactions with vascular endothelial cells initiate the development of rheumatic carditis.

The role of group A streptococcal and Streptococcus dysgalactiae subspecies equisimilis M-protein specific Abs and T-cells in endothelial cell activation was investigated using cultured rat aortic endothelial cells, and in a rat model of autoimmune valvulitis. Heat inactivated serum and mononuclear cells from streptococcal M-protein immunized rats independently induced upregulation of the endothelial cell adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in cultured cells. We also observed T-cell migration across endothelial cell monolayers incubated with serum from M-protein-immunized rats. Furthermore, we observed VCAM-1 and ICAM-1 expression in the myocardium of rats injected with M-protein compared to control animals. These observations support the contention that initial interactions between streptococcal M-protein specific Abs and/or T-cells with the heart endothelium lead to endothelial cell activation followed by transmigration of M-protein specific T-cells into heart tissue leading to an inflammatory process that leads to carditis in rheumatic fever and rheumatic heart disease.

Variations in the secondary structures of PAM proteins influence their binding affinities to human plasminogen

M-proteins (M-Prts) are major virulence determinants of Group A Streptococcus pyogenes (GAS) that are covalently anchored to the cell wall at their conserved COOH-termini while the NH2-terminal regions extend through the capsule into extracellular space. Functional M-Prts are also secreted and/or released from GAS cells where they exist as helical coiled-coil dimers in solution. Certain GAS strains (Pattern D) uniquely express an M-protein (plasminogen-binding group A streptococcal M-protein; PAM) that directly interacts with human plasminogen (hPg), a process strongly implicated in the virulence of these strains. M-Prt expressed by the emm gene is employed to serotype over 250 known strains of GAS, ∼20 of which are hitherto found to express PAMs. We have developed a modular structural model of the PAM dimer that describes the roles of different domains of this protein in various functions. While the helical COOH-terminal domains of PAM are essential for dimerization in solution, regions of its NH2-terminal domains also exhibit a weak potential to dimerize. We find that temperature controls the open (unwound) or closed (wound) states of the functional NH2-terminal domains of PAM. As temperature increases, α-helices are dramatically reduced, which concomitantly destabilizes the helical coiled-coil PAM dimers. PAMs with two a-repeats within the variable NH2-terminal A-domain (class I/III) bind to hPg tightly, but natural PAM isolates with a single a-repeat in this domain (class II) display dramatic changes in hPg binding with temperature. We conclude that coexistence of two a-repeats in PAM is critical to achieve optimal binding to hPg, especially in its monomeric form, at the biologically relevant temperature.

Group A streptococcal M-protein specific antibodies and T-cells drive the pathology observed in the rat autoimmune valvulitis model

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune mediated diseases triggered by group A streptococcal (GAS) infections. Molecular mimicry between GAS M-proteins and host tissue proteins has been proposed as the mechanism that initiates autoreactive immune responses in ARF/RHD. However, the individual role of antibodies and T-cells specific for GAS M-proteins in the pathogenesis of autoimmune carditis remains under-explored. The current study investigated the role of antibodies and T-cells in the development of carditis in the Lewis rat autoimmune valvultis (RAV) model by transferring serum and/or splenic T-cells from rats previously injected with GAS recombinant M5 protein. Here we report that serum antibodies alone and serum plus in vitro expanded rM5-specific T-cells from hyperimmune rats were capable of transferring carditis to naïve syngeneic animals. Moreover, the rats that received combined serum and T-cells developed more severe carditis. Recipient rats developed mitral valvulitis and myocarditis and showed prolongation of P-R intervals in electrocardiography. GAS M5 protein-specific IgG reactivity and T-cell recall response were also demonstrated in recipient rats indicating long-term persistence of antibodies and T-cells following transfer. The results suggest that both anti-GAS M5 antibodies and T-cells have differential propensity to induce autoimmune mediated carditis in syngeneic rats following transfer. The results highlight that antibodies and effector T-cells generated by GAS M protein injection can also independently home into cardiac tissue to cross-react with tissue proteins causing autoimmune mediated immunopathology.

The Quest for Psoriasis Autoantigens: Genetics Meets Immunology in the Melanocyte

The Quest for Psoriasis Autoantigens: Genetics Meets Immunology in the Melanocyte

Variable region in streptococcal M-proteins provides stable binding with host fibrinogen for plasminogen-mediated bacterial invasion

Dimeric M-proteins (M-Prt) in group A Streptococcus pyogenes (GAS) are surface-expressed virulence factors implicated in processes that contribute to the pathogenicity of infection. Sequence analyses of various GAS M-Prts have shown that they contain a highly conserved sortase A-dependent cell wall-anchored C terminus, whereas the surface-exposed N terminus is highly variable, a feature used for identification and serotyping of various GAS strains. This variability also allows for strain-specific responses that suppress host defenses. Previous studies have indeed identified the N-terminal M-Prt B-domain as the site interacting with antiphagocytotic human-host fibrinogen (hFg). Herein, we show that hFg strongly interacts with M-Prts containing highly variable B-domains. We further demonstrate that specific GAS clinical isolates display high affinity for the D-domain of hFg, and this interaction allowed for subsequent surface binding of human-host plasminogen (hPg) to the E-domain of hFg. This GAS surface-bound hPg is then activated by GAS-secreted streptokinase, leading to the generation of an invasive proteolytic bacterial surface. Our results underscore the importance of the human fibrinolytic system in host-pathogen interactions in invasive GAS infections.

Oral immunization of mice with engineered Lactobacillus gasseri NM713 strain expressing Streptococcus pyogenes M6 antigen.

The aim of this in vivo study was to evaluate the effects of a recombinant probiotic strain, Lactobacillus gasseri NM713, which expresses the conserved region of streptococcal M6 protein (CRR6), as an oral vaccine against Streptococcus pyogenes. A dose of 10(9) cells of the recombinant strain in 150 μL PBS buffer was administered orally to a group of mice. One control group received an equivalent dose of Lb. gasseri NM613 (containing the empty plasmid without insert) or and another control group received PBS buffer. Each group contained 30 mice. The immunization protocol was followed on three consecutive days, after which two booster doses were administered at two week intervals. Fecal and serum samples were collected from the mice on Days 18, 32, 46, 58 after the first immunization and Day 0 prior to immunization. Anti-CRR6 IgA and IgG concentrations were measured by ELISA in fecal and sera samples, respectively, to assess immune responses. Vaccination with the recombinant Lb. gasseri NM713 strain induced significant protection after nasal challenge with S. pyogenes, only a small percentage of this group developing streptococcal infection (10%) or dying of it (3.3%) compared with the NM613 and PBS control groups, high percentages of which developed streptococcal infection (43.3% and 46.7%, respectively) and died of it (46.7% and 53%, respectively). These results indicate that recombinant Lb. gasseri NM713 has potential as an oral delivery vaccine against streptococcus group A.

The Inductive Effect of Purified Streptococcal M-protein on Immunity against Streptococcus pyogenes in Rabbit Model

The Inductive Effect of Purified Streptococcal M-protein on Immunity against Streptococcus pyogenes in Rabbit Model

A Case of Acute Rheumatic Fever With Henoch Schonlein Purpura.

Dear Editor, The relation of Henoch-Schonlein purpura (HSP) vasculitis with Acute rheumatic fever was defined by Gairdner firstly in 1948 (1). Both HSP and rheumatic fever can affect the joints and heart in a different way. In HSP artritis is usually non migratory and heart involvement is usually appear as myocarditis. In rheumatic fever artritis is usually migratory and heart involvement is manifest as valvulitis. Rheumatic fever may rarely develop during HSP. The role of group A beta-haemolytic streptococcus (GABHS) in the pathogenesis of rheumatic fever is well established. The presence of rheumatic fever, GABHS may sometimes have a pathogenic role in HSP (2). We present here a child admitted to our clinic as acute rheumatic carditis, who developed HSP vasculitis thereafter. A 12- year-old male presented to our clinic with initially right ankle,followed by left knee and left ankle, pain, sensitivity, redness. His complaints had started 4 days ago. We learned that he had an upper respiratory infection 15 days ago and used antibiotic. At the time of admission axillary body temperature was 36.7°C; blood pressure was 90/60 mmHg. Erythema marginatum-like eruptions appeared on his body and upper extremities, which regressed within 1 hour during follow-up (Figure 1). Figure 1. Marginatum-Like Eruptions on His Body A cardiac auscultation revealed a grade 2/6 systolic murmur over the mitral region. He had hyperemia and swelled on the left knee and right leg, a pronounced diameter difference between the two ankles. Laboratory investigation showed hemoglobin 11.2 g/dL, WBC count 7.6 × 109/L, platelet count 283 × 109/L, C-reactive protein was elevated 79.9 mg/dL, erythrocyte sedimentation rate was elevated, 80 mm/hour. Antistreptolysin (ASO) titer was 180 Todd Units on admission. ASO was elevated 320 Todd Units during follow-up. The throat culture was negative. Antinuclear antibody was absent, rheumatoid factor was negative. Electrocardiography revealed a PR interval of 0.20 seconds. Echocardiographic findings revealed minimal mitral, aortic, and tricuspid valve failure. The patient was diagnosed with ARF carditis, and treatment was started with 2 mg/kg/day prednisolone. Benzathine penicillin made intramuscular. During the second day of hospitalization patient developed purpuric rush on his lower ekstremities. Purpuric rush, did not become pale with pressure. The skin biopsy revealed leucocytoclastic vasculitis (Henoch-Schonlein purpura) (Figure 2). Figure 2. Skin Biopsy Image His serum immune globulin A (IgA) level was elevated 324 mg/dL. Therefore, the steroid treatment was continued. During follow-up the signs and symptoms of vasculitis and carditis regressed. Our patient had migratory artritis. According to the Jones criteria, our case had three major diagnostic symptoms of rheumatic fever; arthritis, carditis, and erythema marginatum; therefore, a diagnosis of acute rheumatic fever was made. ASO, which is a supporting parameter of diasease, was also high in our case. Ocal et al. (3) reported a case of HSP with rheumatic carditis with history of streptococcal infection 4 weeks ago. Our patient had history of upper respiratory infection before 2 weeks ago. Our case’s throat culture was negative for GABHS, but he used antibiotic. Elevated ASO titers suggested a recent streptococcus infection. Immunologic mechanism underlying HSP and rheumatic fever are quite different. Celluler immune mechanism mediated by T lymphocytes to streptococcal M-proteins cause destruction cardiac tissues (4). In these patients elevated IgA levels and immune complexes have been described (5). In previous reports during to HSP, developed acute rheumatic carditis thereafter (1, 3, 6). He is different from previous cases. In literature, the interval from onset of HSP to appearance of rheumatic fever was declared as 10 days to 12 weeks (2, 3, 6). Literatures are limited of HSP and acute rheumatic fever co-occurrence without time interval between these two diseases (6, 7). We presented this case due to that rheumatic fever appeared first, after 2 days purpuric rash occurred. Because of these two reasons our case is different from previous reports. We think that GABHS infections may trigger HSP as acute rheumatic fever.

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein

Streptokinase (SK), secreted by Group A Streptococcus (GAS), is a single-chain ∼47-kDa protein containing three consecutive primary sequence regions that comprise its α, β, and γ modules. Phylogenetic analyses of the variable β-domain sequences from different GAS strains suggest that SKs can be arranged into two clusters, SK1 and SK2, with a subdivision of SK2 into SK2a and SK2b. SK2b is secreted by skin-tropic Pattern D M-protein strains that also express plasminogen (human Pg (hPg)) binding Group A streptococcal M-protein (PAM) as its major cell surface M-protein. SK2a-expressing strains are associated with nasopharynx tropicity, and many of these strains express human fibrinogen (hFg) binding Pattern A-C M-proteins, e.g. M1. PAM interacts with hPg directly, whereas M1 binds to hPg indirectly via M1-bound hFg. Subsequently, SK is secreted by GAS and activates hPg to plasmin (hPm), thus generating a proteolytic surface on GAS that enhances its dissemination. Due to these different modes of hPg/hPm recognition by GAS, full characterizations of the mechanisms of activation of hPg by SK2a and SK2b and their roles in GAS virulence are important topics. To more fully examine these subjects, isogenic chimeric SK- and M-protein-containing GAS strains were generated, and the virulence of these chimeric strains were analyzed in mice. We show that SK and M-protein alterations influenced the virulence of GAS and were associated with the different natures of hPg activation and hPm binding. These studies demonstrate that GAS virulence can be explained by disparate hPg activation by SK2a and SK2b coupled with the coinherited M-proteins of these strains.

Identification of Streptococcal M-Protein Cardiopathogenic Epitopes in Experimental Autoimmune Valvulitis

The M protein of rheumatogenic group A streptococci induces carditis and valvulitis in Lewis rats and may play a role in pathogenesis of rheumatic heart disease. To identify the epitopes of M5 protein that produce valvulitis, synthetic peptides spanning A, B, and C repeat regions contained within the extracellular domain of the streptococcal M5 protein were investigated. A repeat region peptides NT4, NT5/6, and NT7 induced valvulitis similar to the intact pepsin fragment of M5 protein. T cell lines from rats with valvulitis recognized M5 peptides NT5/6 and NT6. Passive transfer of an NT5/6-specific T cell line into naïve rats produced valvulitis characterized by infiltration of CD4+ cells and upregulation of VCAM-1, while an NT6-specific T cell line did not target the valve. Our new data suggests that M protein-specific T cells may be important mediators of valvulitis in the Lewis rat model of rheumatic carditis.

Rheumatic Heart Disease: Key Points on Valve Lesions Development

Rheumatic heart disease lesions are mediated by inflammatory and autoimmune reactions. Several genes related to both innate and adaptive immune responses are involved in the development of the disease. Adhesion molecules and chemokines facilitate heart tissue-T cell infiltration. T lymphocytes are the major effectors of crossreactivity between streptococcal and human proteins leading to RHD. The in vitro analysis of heart-tissue infiltrating T cells showed their ability to recognize streptococcal-M protein peptides as well as self-antigens by molecular mimicry mechanism. A cytokine balance favoring Th1 polarization and production of inflammatory cytokines and few IL-4 producing cells in the valves lead to the progression and maintenance of valvular lesions.

Group A Streptococcal Vaccine Candidates based on the Conserved Conformational Epitope from M Protein

Copper catalysed azide alkyne Huisgen cycloaddition (click) reaction assisted conjugation of the multiple copy of conserve epitope (J14) derived from Group A Streptococcal M-protein to LCP core induced desired α-helical conformation of the epitope. The constructs were able to self assemble into large nanoparticles under aqueous conditions. Immunological assessment of these particles demonstrated their capacity to elicit high-titer systemic IgG antibodies against the epitope without help of adjuvant. The LCP systems incorporating the epitope via its N- or C-termini did not elicit significantly different immune responses. Keywords: Epitope orientation, group A streptococcus, infectious disease, lipid core peptide, nanoparticles, self-adjuvanting, lipopeptide vaccine, N-TERMINUS J14 AZIDE, HUISGEN CYCLOADDITION, LCP CORE

Group A Streptococcal Vaccine Candidates based on the Conserved Conformational Epitope from M Protein

Copper catalysed azide alkyne Huisgen cycloaddition (click) reaction assisted conjugation of the multiple copy of conserve epitope (J14) derived from Group A Streptococcal M-protein to LCP core induced desired α-helical conformation of the epitope. The constructs were able to self assemble into large nanoparticles under aqueous conditions. Immunological assessment of these particles demonstrated their capacity to elicit high-titer systemic IgG antibodies against the epitope without help of adjuvant. The LCP systems incorporating the epitope via its N- or C-termini did not elicit significantly different immune responses. Keywords: Epitope orientation, group A streptococcus, infectious disease, lipid core peptide, nanoparticles, self-adjuvanting, lipopeptide vaccine, N-TERMINUS J14 AZIDE, HUISGEN CYCLOADDITION, LCP CORE

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

BackgroundThe assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.ResultsFragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell.ConclusionsWe report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals.

B- and T-Cell Responses in Group A Streptococcus M-Protein- or Peptide-Induced Experimental Carditis

The etiology of rheumatic fever and rheumatic heart disease (RF/RHD) is believed to be autoimmune, involving immune responses initiated between streptococcal and host tissue proteins through a molecular mimicry mechanism(s). We sought to investigate the humoral and cellular responses elicited in a Lewis rat model of group A streptococcus M-protein- or peptide-induced experimental valvulitis/carditis, a recently developed animal model which may, in part, represent human rheumatic carditis. Recombinant streptococcal M5 protein elicited opsonic antibodies in Lewis rats, and anti-M5 antisera recognized epitopes within the B- and C-repeat regions of M5. One peptide from the streptococcal M5 protein B-repeat region (M5-B.6, amino acids 161 to 180) induced lymphocytes that responded to both recombinant M5 and cardiac myosin. Rats immunized with streptococcal M5 protein developed valvular lesions, distinguished by infiltration of CD3(+), CD4(+), and CD68(+) cells into valve tissue, consistent with human studies that suggest that RF/RHD are mediated by inflammatory CD4(+) T cells and CD68(+) macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD.

Crucial Role of the CB3-Region of Collagen IV in PARF-Induced Acute Rheumatic Fever

Acute rheumatic fever (ARF) and rheumatic heart disease are serious autoimmune sequelae to infections with Streptococcus pyogenes. Streptococcal M-proteins have been implicated in ARF pathogenesis. Their interaction with collagen type IV (CIV) is a triggering step that induces generation of collagen-specific auto-antibodies. Electron microscopy of the protein complex between M-protein type 3 (M3-protein) and CIV identified two prominent binding sites of which one is situated in the CB3-region of CIV. In a radioactive binding assay, M3-protein expressing S. pyogenes and S. gordonii bound the CB3-fragment. Detailed analysis of the interactions by surface plasmon resonance measurements and site directed mutagenesis revealed high affinity interactions with dissociation constants in the nanomolar range that depend on the recently described collagen binding motif of streptococcal M-proteins. Because of its role in the induction of disease-related collagen autoimmunity the motif is referred to as “peptide associated with rheumatic fever” (PARF). Both, sera of mice immunized with M3-protein as well as sera from patients with ARF contained anti-CB3 auto-antibodies, indicating their contribution to ARF pathogenesis. The identification of the CB3-region as a binding partner for PARF directs the further approaches to understand the unusual autoimmune pathogenesis of PARF-dependent ARF and forms a molecular basis for a diagnostic test that detects rheumatogenic streptococci.