Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Jul;64(7):2548–2555. doi: 10.1128/iai.64.7.2548-2555.1996

Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci.

K S McIver 1, S Subbarao 1, E M Kellner 1, A S Heath 1, J R Scott 1
PMCID: PMC174109  PMID: 8698478

Abstract

The protein Mga (mga), which is required for transcription of several virulence genes of group A streptococci (GAS), including the antiphagocytic M protein, was suggested to act as the response regulator element of a bacterial two-component pathway. To investigate whether a gene encoding a cognate sensor protein is located upstream of mga, 3.1 kb of DNA 5' of the mga translational start site was cloned from serotype M6 GAS strain JRS4. Sequence analysis of this region revealed two adjacent open reading frames, a previously described orf and a new locus, isp (immunogenic secreted protein), which could encode proteins of 9 and 59 kDa, respectively. Inactivation of either open reading frame had no significant effect on transcription of the gene encoding M protein (emm) under normal growth conditions, suggesting that neither isp nor orf is involved in the Mga regulatory circuit. A protein migrating at an apparent molecular weight of 65,000 was produced when isp was transcribed and translated in vitro. The predicted isp product (Isp) contains an amino-terminal signal sequence region homologous to that found in bacterial secreted proteins, and expression of isp in Escherichia coli resulted in the presence of Isp in the periplasmic fraction. Convalescent-phase serum from a patient with an active GAS infection recognized forms of Isp both from the periplasm of E. coli and the supernatant of a GAS strain. Both isp and orf are highly conserved among strains of GAS, as shown by hybridization analyses.

Full Text

The Full Text of this article is available as a PDF (488.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albright L. M., Huala E., Ausubel F. M. Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu Rev Genet. 1989;23:311–336. doi: 10.1146/annurev.ge.23.120189.001523. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caparon M. G., Geist R. T., Perez-Casal J., Scott J. R. Environmental regulation of virulence in group A streptococci: transcription of the gene encoding M protein is stimulated by carbon dioxide. J Bacteriol. 1992 Sep;174(17):5693–5701. doi: 10.1128/jb.174.17.5693-5701.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caparon M. G., Scott J. R. Excision and insertion of the conjugative transposon Tn916 involves a novel recombination mechanism. Cell. 1989 Dec 22;59(6):1027–1034. doi: 10.1016/0092-8674(89)90759-9. [DOI] [PubMed] [Google Scholar]
  5. Caparon M. G., Scott J. R. Identification of a gene that regulates expression of M protein, the major virulence determinant of group A streptococci. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8677–8681. doi: 10.1073/pnas.84.23.8677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen C., Bormann N., Cleary P. P. VirR and Mry are homologous trans-acting regulators of M protein and C5a peptidase expression in group A streptococci. Mol Gen Genet. 1993 Dec;241(5-6):685–693. doi: 10.1007/BF00279912. [DOI] [PubMed] [Google Scholar]
  7. Cleary P., Retnoningrum D. Group A streptococcal immunoglobulin-binding proteins: adhesins, molecular mimicry or sensory proteins? Trends Microbiol. 1994 Apr;2(4):131–136. doi: 10.1016/0966-842x(94)90600-9. [DOI] [PubMed] [Google Scholar]
  8. Dybvig K., Hollingshead S. K., Heath D. G., Clewell D. B., Sun F., Woodard A. Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas. J Bacteriol. 1992 Apr;174(8):2729–2732. doi: 10.1128/jb.174.8.2729-2732.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Firth N., Ridgway K. P., Byrne M. E., Fink P. D., Johnson L., Paulsen I. T., Skurray R. A. Analysis of a transfer region from the staphylococcal conjugative plasmid pSK41. Gene. 1993 Dec 22;136(1-2):13–25. doi: 10.1016/0378-1119(93)90442-6. [DOI] [PubMed] [Google Scholar]
  10. Fischetti V. A., Jones K. F., Manjula B. N., Scott J. R. Streptococcal M6 protein expressed in Escherichia coli. Localization, purification, and comparison with streptococcal-derived M protein. J Exp Med. 1984 Apr 1;159(4):1083–1095. doi: 10.1084/jem.159.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
  12. Haanes E. J., Cleary P. P. Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49. J Bacteriol. 1989 Dec;171(12):6397–6408. doi: 10.1128/jb.171.12.6397-6408.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hollingshead S. K., Arnold J., Readdy T. L., Bessen D. E. Molecular evolution of a multigene family in group A streptococci. Mol Biol Evol. 1994 Mar;11(2):208–219. doi: 10.1093/oxfordjournals.molbev.a040103. [DOI] [PubMed] [Google Scholar]
  14. Hynes W. L., Ferretti J. J., Gilmore M. S., Segarra R. A. PCR amplification of streptococcal DNA using crude cell lysates. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):139–142. doi: 10.1016/0378-1097(92)90597-h. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. McIver K. S., Heath A. S., Green B. D., Scott J. R. Specific binding of the activator Mga to promoter sequences of the emm and scpA genes in the group A streptococcus. J Bacteriol. 1995 Nov;177(22):6619–6624. doi: 10.1128/jb.177.22.6619-6624.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McIver K. S., Heath A. S., Scott J. R. Regulation of virulence by environmental signals in group A streptococci: influence of osmolarity, temperature, gas exchange, and iron limitation on emm transcription. Infect Immun. 1995 Nov;63(11):4540–4542. doi: 10.1128/iai.63.11.4540-4542.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morton T. M., Eaton D. M., Johnston J. L., Archer G. L. DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1. J Bacteriol. 1993 Jul;175(14):4436–4447. doi: 10.1128/jb.175.14.4436-4447.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Okada N., Geist R. T., Caparon M. G. Positive transcriptional control of mry regulates virulence in the group A streptococcus. Mol Microbiol. 1993 Mar;7(6):893–903. doi: 10.1111/j.1365-2958.1993.tb01180.x. [DOI] [PubMed] [Google Scholar]
  20. Perez-Casal J., Caparon M. G., Scott J. R. Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol. 1991 Apr;173(8):2617–2624. doi: 10.1128/jb.173.8.2617-2624.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Perez-Casal J., Price J. A., Maguin E., Scott J. R. An M protein with a single C repeat prevents phagocytosis of Streptococcus pyogenes: use of a temperature-sensitive shuttle vector to deliver homologous sequences to the chromosome of S. pyogenes. Mol Microbiol. 1993 May;8(5):809–819. doi: 10.1111/j.1365-2958.1993.tb01628.x. [DOI] [PubMed] [Google Scholar]
  22. Perez-Casal J., Swartley J. S., Scott J. R. Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli. Infect Immun. 1990 Nov;58(11):3594–3600. doi: 10.1128/iai.58.11.3594-3600.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Podbielski A., Flosdorff A., Weber-Heynemann J. The group A streptococcal virR49 gene controls expression of four structural vir regulon genes. Infect Immun. 1995 Jan;63(1):9–20. doi: 10.1128/iai.63.1.9-20.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Podbielski A., Peterson J. A., Cleary P. Surface protein-CAT reporter fusions demonstrate differential gene expression in the vir regulon of Streptococcus pyogenes. Mol Microbiol. 1992 Aug;6(16):2253–2265. doi: 10.1111/j.1365-2958.1992.tb01401.x. [DOI] [PubMed] [Google Scholar]
  25. Podbielski A. Three different types of organization of the vir regulon in group A streptococci. Mol Gen Genet. 1993 Feb;237(1-2):287–300. doi: 10.1007/BF00282810. [DOI] [PubMed] [Google Scholar]
  26. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schneewind O., Model P., Fischetti V. A. Sorting of protein A to the staphylococcal cell wall. Cell. 1992 Jul 24;70(2):267–281. doi: 10.1016/0092-8674(92)90101-h. [DOI] [PubMed] [Google Scholar]
  29. Scott J. R. A new gene controlling lysogeny in phage P1. Virology. 1972 Apr;48(1):282–283. doi: 10.1016/0042-6822(72)90139-0. [DOI] [PubMed] [Google Scholar]
  30. Scott J. R., Guenthner P. C., Malone L. M., Fischetti V. A. Conversion of an M- group A streptococcus to M+ by transfer of a plasmid containing an M6 gene. J Exp Med. 1986 Nov 1;164(5):1641–1651. doi: 10.1084/jem.164.5.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  32. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stevens D. L. Invasive group A streptococcus infections. Clin Infect Dis. 1992 Jan;14(1):2–11. doi: 10.1093/clinids/14.1.2. [DOI] [PubMed] [Google Scholar]
  34. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  35. Whatmore A. M., Kapur V., Sullivan D. J., Musser J. M., Kehoe M. A. Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci. Mol Microbiol. 1994 Nov;14(4):619–631. doi: 10.1111/j.1365-2958.1994.tb01301.x. [DOI] [PubMed] [Google Scholar]
  36. Whatmore A. M., Kehoe M. A. Horizontal gene transfer in the evolution of group A streptococcal emm-like genes: gene mosaics and variation in Vir regulons. Mol Microbiol. 1994 Jan;11(2):363–374. doi: 10.1111/j.1365-2958.1994.tb00316.x. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES