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• W2040437764 abstract "Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different β-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever. Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different β-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever. Acute rheumatic fever (ARF) 3The abbreviations used are: ARF, acute rheumatic fever; FOG, fibrinogen-binding protein of GGS; GAS, group A streptococci; GCS, group C streptococci; GGS, group G streptococci; HA, hyaluronic acid; PARF, peptide associated with rheumatic fever; PBS, phosphate-buffered saline; PBST, PBS/Tween; RHD, rheumatic heart disease; SPR, surface plasmon resonance; THY, Todd Hewitt broth with yeast; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay. is one of the most serious diseases caused by streptococci and occurs as an autoimmune sequela of untreated or inadequately treated group A streptococcal pharyngitis (1Bisno A.L. N. Engl. J. Med. 1991; 325: 783-793Crossref PubMed Scopus (381) Google Scholar). ARF and the subsequent rheumatic heart disease (RHD) remain significant causes of cardiovascular disease today (2World Health Organization Rheumatic Fever and Rheumatic Heart Disease: Report of a WHO Expert Consultation, WHO Technical Report Series 923. World Health Organization, Geneva, Switzerland2004Google Scholar, 3Carapetis J.R. McDonald M. Wilson N.J. Lancet. 2005; 366: 155-168Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). The most devastating effects are on children and young adults in their most productive years (2World Health Organization Rheumatic Fever and Rheumatic Heart Disease: Report of a WHO Expert Consultation, WHO Technical Report Series 923. World Health Organization, Geneva, Switzerland2004Google Scholar, 3Carapetis J.R. McDonald M. Wilson N.J. Lancet. 2005; 366: 155-168Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 4Kumar R. Raizada A. Aggarwal A.K. Ganguly N.K. Indian Heart J. 2002; 54: 54-58PubMed Google Scholar). According to a recent estimate more than 15 million people have RHD, more than 0.5 million acquire ARF each year, and about 0.25 million deaths annually are directly attributable to ARF or RHD (2World Health Organization Rheumatic Fever and Rheumatic Heart Disease: Report of a WHO Expert Consultation, WHO Technical Report Series 923. World Health Organization, Geneva, Switzerland2004Google Scholar). The fact that penicillin has clearly failed to eradicate ARF and that streptococcal vaccines are still years away from being available underlines the need for novel control strategies (5Kaplan E.L. Heart. 2005; 91: 3-4Crossref PubMed Scopus (31) Google Scholar, 6Saxena A. Curr. Treat. Options Cardiovasc. Med. 2002; 4: 309-319Crossref PubMed Scopus (13) Google Scholar). Identification of the pathogenic mechanisms underlying ARF is a prerequisite for the development of the necessary diagnostic and preventive approaches. Major virulence factors of streptococci that infect humans are the M and M-like proteins. They exert anti-phagocytic effects (7Bisno A.L. Brito M.O. Collins C.M. Lancet Infect. Dis. 2003; 3: 191-200Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 8Cunningham M.W. Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1769) Google Scholar, 9Fischetti V.A. Clin. Microbiol. Rev. 1989; 2: 285-314Crossref PubMed Scopus (638) Google Scholar, 10Lancefield R.C. J. Immunol. 1962; 89: 307-313Crossref PubMed Google Scholar) and facilitate streptococcal survival within polymorph nuclear neutrophils (11Staali L. Mörgelin M. Björck L. Tapper H. Cell. Microbiol. 2003; 5: 253-265Crossref PubMed Scopus (71) Google Scholar). Variability in the N-terminal of M proteins generated more than 100 distinct M serotypes. Rheumatogenicity of Streptococcus pyogenes strains, which are also referred to as group A streptococci (GAS), was found to correlate with certain M serotypes in different parts of the world (12Potter E.V. Svartman M. Mohammed I. Cox R. Poon-King T. Earle D.P. J. Pediatr. 1978; 92: 325-333Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 13Majeed H.A. Khuffash F.A. Yousof A.M. Farwana S.S. Chugh T.D. Moussa M.A. Rotta J. Havlickova H. Zentralbl. Bakteriol. Mikrobiol. Hyg. A. 1986; 262: 346-356PubMed Google Scholar, 14Stollerman G.H. Adv. Intern. Med. 1990; 35: 1-25PubMed Google Scholar), indicating that such M proteins may be directly involved in the pathogenesis of ARF/RHD. Different autoimmune mechanisms have been proposed for pathogenesis of ARF (3Carapetis J.R. McDonald M. Wilson N.J. Lancet. 2005; 366: 155-168Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 8Cunningham M.W. Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1769) Google Scholar, 15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar) that bases on M proteins and other surface components of GAS as causative agents and results in autoimmune response against host proteins of cardiac tissues (3Carapetis J.R. McDonald M. Wilson N.J. Lancet. 2005; 366: 155-168Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 4Kumar R. Raizada A. Aggarwal A.K. Ganguly N.K. Indian Heart J. 2002; 54: 54-58PubMed Google Scholar, 8Cunningham M.W. Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1769) Google Scholar, 15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar, 16Tontsch D. Pankuweit S. Maisch B. Clin. Exp. Immunol. 2000; 121: 270-274Crossref PubMed Scopus (24) Google Scholar, 17Ellis N.M. Li Y. Hildebrand W. Fischetti V.A. Cunningham M.W. J. Immunol. 2005; 175: 5448-5456Crossref PubMed Scopus (93) Google Scholar). One recent hypothesis is that human collagen IV, a major component of subendothelial basement membranes (18Afek A. Shoenfeld Y. Manor R. Goldberg I. Ziporen L. George J. Polak-Charcon S. Amigo M.C. Garcia-Torres R. Segal R. Kopolovic J. Lupus. 1999; 8: 502-507Crossref PubMed Scopus (59) Google Scholar, 19Yurchenco P.D. Schittny J.C. FASEB J. 1990; 4: 1577-1590Crossref PubMed Scopus (790) Google Scholar), acts as such an autoantigen after forming a complex with S. pyogenes strains, which have a potential of causing ARF (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar). Investigations on S. pyogenes strains of the rheumatogenic M serotypes M3 and M18 identified both M3 protein of the M3 strain and the hyaluronic acid (HA) capsule of the M18 strain as streptococcal surface components that interact with collagen. Immunization of mice with M3 protein led to the generation of collagen IV autoantibodies, which were also found in sera of patients with ARF or RHD. Direct binding of collagen to streptococcal surface components is, therefore, considered as one of the key mechanisms for induction of ARF (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar). In geographical areas with a high incidence of ARF there is a widespread carriage of group C streptococci (GCS) and group G streptococci (GGS) (20Haidan A. Talay S.R. Rohde M. Sriprakash K.S. Currie B.J. Chhatwal G.S. Lancet. 2000; 356: 1167-1169Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 21McDonald M.I. Towers R.J. Andrews R.M. Benger N. Currie B.J. Carapetis J.R. Clin. Infect. Dis. 2006; 43: 683-689Crossref PubMed Scopus (135) Google Scholar, 22Ahmed J. Zaman M.M. Keramat Ali S.M. Bangladesh Med. Res. Council Bull. 2003; 29: 113-117PubMed Google Scholar). Traditionally, these serogroups are considered as important veterinary pathogens but are also involved in human infections. Their spectrum of diseases is very similar to the one of GAS, comprising pharyngitis, skin, and soft tissue infections as well as bacteremia and streptococcal toxic shock syndrome (23Baracco G.J. Bisno A.L. Fischetti V.A. Gram-positive Pathogens. 2nd Ed. ASM Press, Washington, D. C.2006: 223-229Google Scholar). Some GCS and GGS strains have the potential to induce autoimmunity against cardiac myosin, suggesting contribution to the pathogenesis of ARF/RHD (20Haidan A. Talay S.R. Rohde M. Sriprakash K.S. Currie B.J. Chhatwal G.S. Lancet. 2000; 356: 1167-1169Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). This might at least partially explain the high incidence of ARF in certain indigenous populations of the Northern Territory of Australia, where pharyngeal isolation of GAS is rare but the prevalence of GCS and GGS is high (24McDonald M. Currie B.J. Carapetis J.R. Lancet Infect. Dis. 2004; 4: 240-245Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The mechanisms known today are not sufficient to fully explain the pathogenesis of ARF. In this study we investigated the potential of GCS and GGS to interact with collagen IV and identified the binding factors, one being the M-like protein FOG, which was described as an adhesin (25Nitsche D.P. Johansson H.M. Frick I.M. Mörgelin M. J. Biol. Chem. 2006; 281: 1670-1679Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Analysis of patient sera revealed contribution of FOG in clinical cases of ARF/RHD, indicating the role of GGS as a causative organism. By pinpointing the responsible binding motif we could gain deeper insight into the pathogenesis of ARF and discovered a promising diagnostic marker for the recognition of rheumatogenic strains. Bacterial Strains and Human Sera—Group C and G streptococcal strains were collected in different geographic regions, with an emphasis on human isolates from the Northern Territory in Australia. The S. pyogenes control strain (A60) was obtained from the Institute for Medical Microbiology of the TU Aachen. Unless stated otherwise, streptococci were grown statically to late exponential phase in tryptic soy broth (Roth) at 37 °C. Human sera from patients with acute rheumatic fever and of healthy volunteers were collected in Chandigarh, India. Collagen Binding Assays—Streptococci were suspended in phosphate-buffered saline (PBS) to give 108 bacteria/ml. Bacteria (2.5 × 107) were incubated with 30 ng (100,000 cpm) of 125I-labeled collagen IV isolated from placenta (Sigma) for 45 min at room temperature. Bacteria were harvested by centrifugation, washed in PBS with 0.05% Tween 20 (PBST), and the pellet was measured in a gamma counter. The HA capsule was detected by preincubating bacteria with either 20 μl (60 units) of hyaluronidase (Applichem, Darmstadt, Germany) or 20 μl of PBS for 45 min at 37 °C. The bacteria were washed 3 times in 1 ml of PBS before the incubation with the radiolabeled collagen. Assays were performed in triplicate within each set of experiments, and experiments were repeated on different days. Screening and Sequencing of emm and emm-like Genes—The chromosomal DNA of all collagen binding isolates was tested by PCR for the presence of emm and emm-like genes using the primers 1 and 2 recommended by the Center of Disease Control. The obtained PCR products were subsequently sequenced using primer 1. Electron Microscopy—G45 (a FOG-positive, HA-negative, and collagen binding strain) and G50 (a non-collagen binding strain) were grown statically to late exponential phase in Todd Hewitt broth supplemented with 0.5% yeast extract (THY). The bacteria (5 × 107) were suspended in 500 μl of PBS and incubated with 40 μg of collagen IV at room temperature for 30 min. Samples were washed, fixed, and processed for field emission scanning electron microscopy as described before (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar). Recombinant Proteins and Site-directed Mutagenesis—For cloning of FOG constructs and of MLC1903, suitable PCR products were cloned into the pGEX-6P-1 vector (GE Healthcare) between the BamHI and the SalI or the BamHI and the EcoRI cleavage site. FOGfl (amino acid residues 1-557) represents the mature full-length protein. FOG1-B represents the first 278 amino acids, and FOG1-A represents the first 134 amino acids of the mature FOG protein (Fig. 2A). The plasmids coded for fusion proteins that carried an N-terminal GST tag. They were expressed in Escherichia coli HB101. Site-directed mutagenesis was performed on the plasmid pGEX-6P-1-FOGfl that coded for the GST-FOGfl fusion protein. E. coli clones containing the plasmids with the desired substitutions were generated using the GeneTailor™ site-directed mutagenesis system (Invitrogen) following the manufacturer's recommendations. The tagged proteins from E. coli lysates were bound to glutathione-Sepharose 4B (GE Healthcare) and eluted with 10 mm reduced l-glutathione (Sigma) in 50 mm Tris-HCl (pH 8.0) after washing the matrix with PBS. When desired the GST tag was removed by digesting the proteins with PreScission™ protease (GE Healthcare) while bound to the affinity matrix, which eluted the untagged FOG proteins. Immunization of Mice—For immunization with FOGfl and FOGB2-C2, groups of pathogen-free 8-week-old female C3H mice were injected intraperitoneally with 200 μl of an emulsion of the appropriate recombinant protein (10 μg) in PBS (2/3) and Freund's incomplete adjuvant (1/3) at days 1, 14, and 21. Emulsion devoid of FOG was injected into mice of the control group. Serum samples were taken at day 28. In experiments with M3.5 and FOG1-B, respectively, groups of five pathogen-free 8-week-old female BALB/c mice were immunized intraperitoneally with an emulsion of 25 μg of the regarding recombinant protein in 50 μl of PBS and 50 μl of Freund's incomplete adjuvant per dose at days 1, 7, and 14. Control mice were injected with 50 μl of PBS and 50 μl of Freund's incomplete adjuvant per dose. At day 21, the serum samples were collected and tested in enzyme-linked immunosorbent assay (ELISA) as described below. To absorb FOG-reactive antibodies, 10 μl of serum pool was diluted 1:50 in PBS and incubated for 1 h with 1 mg of GST-FOGfl protein immobilized on glutathione-Sepharose 4B. Antibodies that bound to this affinity matrix were removed by centrifugation. The supernatant was subjected to ELISA analysis. ELISA—To determine anti-collagen IV antibody titers, 96-well plates (Greiner, Frickenhausen, Germany) were coated overnight at 4 °C with anti-human collagen IV rabbit polyclonal antibody (Progen, Heidelberg, Germany) diluted 1:100 in 0.1 m NaHCO3 (pH 9.6), blocked with 2% bovine serum albumin in PBST, and incubated with collagen IV (2 μg/ml in PBS) for 1 h at 37 °C. After washing with PBST, mouse sera or human sera diluted 1:50, 1:158, 1:500, and 1:1580 in PBS were added to the wells and incubated overnight at 4 °C. After washing, bound antibodies were detected using suitable horseradish peroxidase-coupled secondary antiserum (goat anti-mouse IgG and IgM, Jackson Laboratories; rabbit anti-human IgG, IgA, and IgM, Sigma-Aldrich) and 2,2-azino-di-[3-ethylbenzthiazoline sulfonate] diammonium salt (ABTS tablets, Roche Applied Science) as substrate. The absorbance was determined at 405 nm. To determine anti-M3 or anti-FOG antibody titers, M3.5 or FOG1-A (4 μg/ml in 0.1 m NaHCO3, pH 9.6) were immobilized overnight at 4 °C followed by blocking with 1% bovine serum albumin in PBST. Wells were washed before human sera diluted 1:100, 1:316, 1:1000, and 1:3162 in 1% bovine serum albumin in PBS were added to the wells and incubated for 1.5 h at 37 °C. Antibody binding was measured as described above. Sera from symptom-free donors with low titers against group A carbohydrate, group G carbohydrate, and streptolysin O were identified. The highest titer against FOG1-A or M3.5, respectively, found in those sera was defined as the cut-off for considering a patient serum positive. Dot Blots—Ligand overlay assays were performed by spotting the purified recombinant proteins (5, 1, or 0.5 μg) on nitrocellulose. The membrane was blocked for 1 h in PBS containing 5% skimmed milk followed by a 1-h incubation with radiolabeled collagen IV (200,000 cpm) in PBST. After five washing steps in PBST, filters were dried and placed on radiographic films (Eastman Kodak Co.) for autoradiography. Surface Plasmon Resonance (SPR)—Protein interactions were studied in a BIAcore 2000 system (BIAcore AB) using 10 mm HEPES, 100 mm NaCl (pH 7.4) as running buffer. A CM5 sensor chip was activated by a 4-min injection of 0.05 m N-hydroxysuccinimide, 0.2 m N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride in water. Collagen IV from human placenta (Sigma, 1 mg/ml in 0.1 m sodium acetate) was diluted 1:25 in 10 mm sodium acetate (pH 5.2). Injection of 3 μl at a flow rate of 5 μl/min led to immobilization of 400–550 response units of collagen IV. Residual reactive groups were inactivated by a 6-min injection of 1 m ethanolamine, 0.1 m NaHCO3, 0.5 m NaCl, 5 mm EDTA (pH 8.0). Collagen I was immobilized as described previously (25Nitsche D.P. Johansson H.M. Frick I.M. Mörgelin M. J. Biol. Chem. 2006; 281: 1670-1679Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Interaction measurements with FOG1-A and FOG1-B were carried out at a flow rate of 60 μl/min measurements with GST-FOGfl, mutated GST-FOGfl, and GST at a flow rate of 35 μl/min. Surface regeneration was achieved by injection of two 30-s pulses of 0.2% SDS in water. The BIAevaluation 3.0 software was used for further analysis of the data. The curves shown represent the difference between the signal of the collagen-coupled surface and of a deactivated control surface devoid of protein. They were further corrected by subtraction of the curve that was obtained after injection of buffer alone. Buffer injection led to responses less than five response units. Spot-synthesized Peptides—The 15-mer peptides were synthesized on an aminopegylated cellulose membrane (AIMS Scientific Products) as described previously (26Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (927) Google Scholar). After washing in ethanol (96%) and thereafter in PBS, the membrane was incubated for 1 h with blocking buffer (2% blocking solution from Genosys and 0.5% sucrose in PBST) at room temperature. Binding was tested by incubating the membrane with 3 μg of radiolabeled collagen IV (1,000,000 cpm) in PBST for 4 h at room temperature. Non-bound ligand was removed by washing with PBST for 1 h. Bound collagen IV was detected using an x-ray film. Collagen Binding Factors on GCS and GGS Isolated from Human Infections—A total of 70 GCS and GGS strains isolated from human infections, all belonging to the species Streptococcus dysgalactiae subsp. equisimilis, were examined in this study. A S. pyogenes strain (A60) of the rheumatogenic M3 serotype was included as a positive control (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar). All strains were tested for binding to radiolabeled collagen IV, revealing that 27 strains (38%) of the isolates had an ability to bind collagen IV, similar to the control strain. All strains that interacted with collagen were subjected to further analyses. Because HA capsule and M3 protein have been reported as collagen binding factors of GAS (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar), the presence of such factors in the GCS and GGS strains was investigated (Table 1). Collagen binding via the HA capsule was tested by hyaluronidase treatment that abolished HA-mediated interaction. HA was found in seven of the isolates and was distributed equally among GGS and GCS. The presence of emm-related genes, coding for M-like proteins, was investigated by PCR using primers of highly conserved sequences (see “Experimental Procedures”) and subsequent DNA sequencing of the PCR products. All isolates possessed emm-related genes, which is in accordance with the observation that the presence of such genes is frequent in human isolates (27Simpson W.J. Robbins J.C. Cleary P.P. Microb. Pathog. 1987; 3: 339-350Crossref PubMed Scopus (32) Google Scholar). Four of the strains were found to possess the emm-related gene fog that codes for the M-like fibrinogen binding protein of GGS (FOG) (25Nitsche D.P. Johansson H.M. Frick I.M. Mörgelin M. J. Biol. Chem. 2006; 281: 1670-1679Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 28Johansson H.M. Mörgelin M. Frick I.M. Microbiology. 2004; 150: 4211-4221Crossref PubMed Scopus (22) Google Scholar). Because FOG was previously described as an adhesin that binds collagen I (25Nitsche D.P. Johansson H.M. Frick I.M. Mörgelin M. J. Biol. Chem. 2006; 281: 1670-1679Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), these strains became the subject of further investigations. The gene fog was present in GGS isolates only. Of the four strains that were found to possess fog, two had an HA capsule that contributed to collagen binding. The interaction of streptococci with collagen IV was also analyzed using field emission scanning electron microscopy (Fig. 1), revealing that a FOG-positive human isolate negative for HA bound collagen IV and aggregated it on its surface (Fig. 1A). Taken together, the experiments allowed the conclusion that encapsulated GCS and GGS bind collagen IV via HA, but direct interaction between M-like protein FOG and collagen IV remained to be investigated.TABLE 1Collagen binding factors in human GCS and GGS isolatesGroupStrain no.Collagen binding factorsemmfogHAC62+--C63+--C72+--C77+--C78+--C82+-+C89+-+G22+--G23+--G24+--G25+--G27+--G33+--G35+--G40+--G41+--G42+--G43+++G45++-G48+--G51++-G52+--G57+-+G58+-+G59+--G60+-+G89+++ Open table in a new tab The N Terminus of FOG Binds Collagen IV—For investigation of the interaction between FOG and collagen IV, three different recombinant proteins, FOGfl (amino acids 1–557), FOG1-B (amino acids 1–278), and FOG1-A (amino acids 1–134) (Fig. 2A), were subjected to SPR measurements using collagen IV as the immobilized ligand. FOGfl (Fig. 2B) as well as the shorter fragments FOG1-B (Fig. 2C) and FOG1-A (Fig. 2D) showed concentration-dependent binding with dissociation constants of 6 nm, 53 nm, and 1.3 μm, respectively. Coherent with data on collagen I (25Nitsche D.P. Johansson H.M. Frick I.M. Mörgelin M. J. Biol. Chem. 2006; 281: 1670-1679Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), these experiments indicated that the N-terminal serotype-specific region of FOG is a major collagen binding factor found in GGS isolates from human infections. Binding of FOG to collagen IV, which was shown here for the first time, is an interaction that suggests a role of FOG in ARF pathogenesis (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar). Binding of FOG to collagen I (25Nitsche D.P. Johansson H.M. Frick I.M. Mörgelin M. J. Biol. Chem. 2006; 281: 1670-1679Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) indicates a low selectivity within the collagen family. This is corroborated by the observation that FOG also binds to collagen type II and type III. 4D. P. Nitsche-Schmitz, unpublished data. The specificity for collagens, however, is proven by the inability of FOG to bind C1q (37Nitsche-Schmitz D.P. Johansson H.M. Sastalla I. Reissmann S. Frick I-M. Chhatwal G.S. J. Biol. Chem. 2007; 282: 17530-17536Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), which is a serum protein with an extensive collagenous moiety (30Reid K.B. Porter R.R. Biochem. J. 1976; 155: 19-23Crossref PubMed Scopus (242) Google Scholar). Moreover, FOG was found negative for binding to fibronectin (data not shown), which is, like collagens, another abundant protein of the extracellular matrix (31Mosher D.F. Annu. Rev. Med. 1984; 35: 561-575Crossref PubMed Scopus (262) Google Scholar). The N Termini of FOG and M3 Induce Collagen Autoimmunity—In the case of GAS the surface protein M3 binds and aggregates collagen, leading to collagen IV autoantigenicity (15Dinkla K. Rohde M. Jansen W.T. Kaplan E.L. Chhatwal G.S. Talay S.R. J. Clin. Investig. 2003; 111: 1905-1912Crossref PubMed Scopus (82) Google Scholar). We, therefore, immunized mice with FOGfl and a FOG fragment that lacks the collagen binding region (FOGB2-C2; amino acids 187–415). Analysis of anti-collagen IV serum titers by ELISA revealed significantly higher autoimmunity in the group immunized with FOGfl as compared with the control group immunized with PBS. Immunization with FOGB2-C2 led to serum titers at the level of the control group (Fig. 3A). The results suggested that the collagen binding N-terminal region of FOG is crucial for the induction of collagen autoimmunity. To test whether the N-terminal fragments of M3 protein or FOG were sufficient for induction of collagen IV autoimmune response, sera from mice immunized with M3.5 (amino acids 1–186) protein or FOG1-B (Fig. 3B), were analyzed. As compared with the buffer control, both groups produced substantially higher titers of anti-collagen IV antibodies. To examine the specificity of the antibodies, the collagen-IV-reactive mouse sera were pooled, and the antibody titers against collagen IV and FOG1-A, determined before and after pre-absorption with FOGfl protein, were compared. Although reactivity against FOG1-A was completely eliminated (Fig. 3C), the serum titer against collagen IV remained almost unchanged after pre-absorption (Fig. 3D), indicating that antibodies against FOG and collagen were distinct populations that did not cross-react. This demonstrated that the observed collagen autoimmunity was not caused by structural similarities of FOG (molecular mimicry) but that interaction with FOG had rendered host collagen IV an antigen. In summary, the experiments indicate that collagen binding motifs in the N-terminal serotype-specific region of FOG and M3 induce collagen IV autoimmunity. To examine whether FOG-collagen complexes induce autoantigenicity in proven clinical cases of ARF/RHD, patient sera with elevated levels of collagen autoantibodies and sera of healthy controls were tested for reactivity against the serotype-specific N terminus of FOG (FOG1-A) as well as M3 protein (M3.5). Substantially higher serum titers against FOG1-A were observed with patient sera as compared with control sera from healthy donors from the same geographic region in Chandigarh, India (Fig. 4). Of the 17 patients' sera examined, 12 sera had positive titers against FOG1-A (for the definition of positive titers, see “Experimental Procedures”). Of these 12, only 5 sera also had a positive titer against M3.5. Notably, sera reacting with M3.5 but not with FOG1-A were not found. The results indicate an epidemiologically important contribution of FOG-positive strains in ARF" @default.
• W2040437764 created "2016-06-24" @default.
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• W2040437764 date "2007-06-01" @default.
• W2040437764 modified "2023-09-29" @default.
• W2040437764 title "Identification of a Streptococcal Octapeptide Motif Involved in Acute Rheumatic Fever" @default.
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