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Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci

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Abstract

Evaluation of homologous regions of published M protein (emm) gene sequences from group A streptococci (GAS; Streptococcus pyogenes) was used to design three primer pairs for polymerase chain reaction (PCR) and three oligonucleotide probe sequences internal to the amplified products. One set of primers and corresponding probe should detect and lead to amplification of emm(-like) genes of virtually every type (“all M”), another (“SOR-M”) should only amplify emm(-like) genes from GAS negative for serum opacity reaction (SOR) and the third (“SOR+M”) should expand only emm(-like) genes from SOR+ GAS. Using the “all M” primer pair for PCR on the genomic DNA from GAS of 29 different M types as well as from a group C and a group G streptococcal isolate, DNA fragments within the expected size range were amplified in every assay. All PCR products reacted with the “all M” probe. Related sequences were not detected in genomic DNA of an S. agalactiae and an Enterococcus faecalis isolate. Applying the “SOR-M” and “SOR+M” primers to identical assays led to mutually exclusive amplification products. The “SOR+M” and “SOR+M” probes hybridized only to their corresponding products. Exceptions to this exclusivity were the SOR+ GAS of M types 3, 8, 27, 34, 42, 67, and 69, which consistently reacted only with the “SOR+M” primer/probe set. Analysis of sequence data from the amplified emm(-like) 2, 3, 18, and 19 genes revealed interesting specific features such as conserved gaps in the C-terminal sequence regions from SOR+ and the exceptional SOR- GAS strains. These data indicate the existence of a subgroup of strains among SOR- GAS and may advance our understanding of phylogenetic relationship between different serotypes of GAS.

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References

  1. Bessen D, Fischetti VA (1990) A human IgG receptor of group A streptococci is associated with tissue site of infection and streptococcal class. J Infect Dis 161:747–754

    Google Scholar 

  2. Bessen D, Jones KF, Fischetti VA (1989) Evidence for two distinct classes of streptococcal M protein and their relationship to rheumatic fever. J Exp Med 169:269–283

    Google Scholar 

  3. Birnboim HC (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol 100:243–255

    Google Scholar 

  4. Chen CC, Cleary PP (1990) Complete nucleotide sequence of the streptococcal C5 a peptidase gene of Streptococcus pyogenes. J Biol Chem 265:3161–3167

    Google Scholar 

  5. Facklam RR, Edwards LR (1979) A reference laboratory's investigations of proposed M-type strains of Streptococcus pyogenes, capsular types of Streptococcus agalactiae, and new group antigens of streptococci. In: Parker MT (ed) Pathogenic Streptococci. Reedbooks, Chertsey, pp 251–253

    Google Scholar 

  6. Fischetti VA (1989) Streptococcal M protein: molecular design and biological behavior. Clin Microbiol Rev 2:285–314

    Google Scholar 

  7. Fischetti VA, Pancholi V, Schneewind O (1990) Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive cocci. Mol Microbiol 4:1603–1605

    Google Scholar 

  8. Frithz E, Heden LO, Lindahl G (1989) Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes. Mol Microbiol 3:1111–1119

    Google Scholar 

  9. Gaworzewska E, Colman G (1988) Changes in pattern of infection caused by Streptococcus pyogenes. Epidemiol Infect 100:257–269

    Google Scholar 

  10. Gomi H, Hozumi T, Hattori S, Tagawa C, Kishimoto F, Björck L (1990) The gene sequence and some properties of protein H. J Immunol 144:4046–4052

    Google Scholar 

  11. Haanes EJ, Cleary PP (1989) Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49. J Bacteriol 171:6397–6408

    Google Scholar 

  12. Haanes-Fritz E, Kraus W, Burdett V, Dale JB, Beachey EH, Cleary P (1988) Comparison of the leader sequences of four group A streptococcal M protein genes. Nucleic Acids Res 16:4667–4677

    Google Scholar 

  13. Heath DG, Cleary PP (1989) Fc-receptor and M-protein genes of group A streptococci are products of gene duplication. Proc Natl Acad Sci USA 86:4741–4745

    Google Scholar 

  14. Hollingshead SK, Fischetti VA, Scott JR (1986) Complete nucleotide sequence of type 6 M protein of the group A streptococcus. J Biol Chem 261:1677–1686

    Google Scholar 

  15. Hollingshead SK, Fischetti VA, Scott JR (1987) Size variation in group A streptococcal M proteins is generated by homologous recombination between intragenic repeats. Mol Gen Genet 207:196–203

    Google Scholar 

  16. Huang TT, Malke H, Ferretti JJ (1989) Heterogeneity of the streptokinase gene in group A streptococci. Infect Immun 57:502–506

    Google Scholar 

  17. Kaplan EL, Johnson DR, Cleary PP (1989) Group A streptococcal serotypes isolated from patients and sibling contacts during the resurgence of rheumatic fever in the United States in the mid-1980's. J Infect Dis 159:101–103

    Google Scholar 

  18. Kusukawa N, Uemori T, Asada K, Kato T (1990) Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. Bio Techniques 9:66–72

    Google Scholar 

  19. Lindahl G (1989) Cell surface proteins of a group A streptococcus type M 4: the IgA receptor and a receptor related to M proteins are coded for by closely linked genes. Mol Gen Genet 216:372–379

    Google Scholar 

  20. Maxted WR, Widdowson JP, Fraser CAM, Ball LC, Bassett DJC (1973) The use of the serum opacity reaction in the typing of group A streptococci. J Med Microbiol 6:83–90

    Google Scholar 

  21. Miller L, Burdett V, Poirier TP, Gray LD, Beachey EH, Kehoe MA (1988) Conservation of protective and nonprotective epitopes in M proteins of group A streptococci. Infect Immun 56:2198–2204

    Google Scholar 

  22. Miller L, Gray L, Beachey E, Kehoe M (1988) Antigenic variation among group A streptococcal M proteins. J Biol Chem 263:5668–5673

    Google Scholar 

  23. Mouw AR, Beachey EH, Burdett V (1988) Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M protein gene and relation to other genes of Streptococcus pyogenes. J Bacteriol 170:676–684

    Google Scholar 

  24. Podbielski A, Kühnemund O, Lütticken R (1990) Identification of group A type 1 streptococcal M protein gene by a non-radioactive oligonucleotide detection method. Med Microbiol Immunol 179:255–262

    Google Scholar 

  25. Relf WA, Sriprakash KS (1990) Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes. FEMS Microbiol Lett 71:345–350

    Google Scholar 

  26. Robbins JC, Spanier JG, Jones SJ, Simpson WJ, Cleary PP (1987) Streptococcus pyogenes type 12 M protein gene regulation by upstream sequences. J Bacteriol 169:5633–5640

    Google Scholar 

  27. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mulis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

    CAS  PubMed  Google Scholar 

  28. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  29. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    CAS  PubMed  Google Scholar 

  30. Saravani GA, Martin DR (1990) Characterization of opacity factor from group-A streptococci. J Med Microbiol 33:55–60

    Google Scholar 

  31. Scott JR, Pulliam WM, Hollingshead SK, Fischetti VA (1985) Relationship of M protein genes in group A streptococci. Proc Natl Acad Sci USA 82:1822–1826

    Google Scholar 

  32. Simpson WJ, Cleary PP (1987) Expression of M type 12 protein by a group A streptococcus exhibits phaselike variation: evidence for coregulation of colony opacity determinants and M protein. Infect Immun 55:2448–2455

    Google Scholar 

  33. Simpson WJ, Robbins JC, Cleary PP (1987) Evidence for group A-related M protein genes in human but not animal-associated group G streptococcal pathogens. Microb Pathogen 3:339–350

    Google Scholar 

  34. Spratt BG, Hedge PJ, Te Heesen S, Edelmann A, Broome-Smith JK (1986) Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 4:337–342

    Google Scholar 

  35. Wilks AF (1989) Cloning members of gene families: an application of the polymerase chain reaction. Technique 1:66–75

    Google Scholar 

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Author notes

  1. Andreas Podbielski

    Present address: Department of Microbiology, UMHC Mayo Building, 420 Delaware St. S. E., Box 196, 55455, Minneapolis, MN, USA

Authors and Affiliations

  1. Institute of Medical Microbiology, Technical University (RWTH), Pauwelsstrasse 30, W-5100, Aachen, Federal Republic of Germany

    Andreas Podbielski, Beate Melzer & Rudolf Lütticken

Authors
  1. Andreas Podbielski
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  2. Beate Melzer
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  3. Rudolf Lütticken
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Podbielski, A., Melzer, B. & Lütticken, R. Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci. Med Microbiol Immunol 180, 213–227 (1991). https://doi.org/10.1007/BF00215250

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  • DOI: https://doi.org/10.1007/BF00215250

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