1887

Microbiology Society logo

Volume 144, Issue 7

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae Free

Abstract

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): adhesin , antigenic size variation , Mycoplasma hyopneumoniae and pathogenesis
© Society for General Microbiology 1998
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-144-7-1931
1998-07-01
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/144/7/mic-144-7-1931.html?itemId=/content/journal/micro/10.1099/00221287-144-7-1931&mimeType=html&fmt=ahah

References

  1. Adzhubei A.A., Sternberg M.J.E. 1993; Left-handed poly-proline II helices commonly occur in globular proteins.. J Mol Biol 229:472–493
     [Google Scholar]
  2. Albertini A.M., Hofer M., Calos P., Miller J.H. 1982; On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions.. Cell 29:319–328
     [Google Scholar]
  3. Avakian A.P., Kleven S.H., Ley D.H. 1991; Comparison of Mycoplasma gallisepticum strains and identification of immunogenic integral membrane proteins with Triton X-114 by immunoblotting.. Vet Microbiol 29:319–328
     [Google Scholar]
  4. Burnette W.N. 1981; Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.. Anal Biochem 112:195–203
     [Google Scholar]
  5. Clark L.K., Armstrong C.H., Freeman M.J., Scheldt A.B., Sands-Freeman L., Knox K. 1991; Investigating the transmission of Mycoplasma hyopneumoniae in a swine herd with enzootic pneumonia.. Vet Med 86:543–550
     [Google Scholar]
  6. Collier A.M., Hu P.C., Clyde W.A. 1985; The changing pathogenicity of Mycoplasma pneumoniae with passage in vitro correlates with virulence.. Diagn Microbiol Infect Dis 3:321–328
     [Google Scholar]
  7. DeBey M.C., Ross R.F. 1994; Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures.. Infect Immun 62:5312–5318
     [Google Scholar]
  8. Djordjevic S.P., Eamens G.J., Romalis L.F., Saunders M.M. 1994; An improved enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies against Mycoplasma hyopneumoniae.. Vet Microbiol 39:261–274
     [Google Scholar]
  9. Djordjevic S.P., Eamens G.J., Romalis L.F., Nicholls P.J., Taylor V., Chin J. 1997; Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant.. Aust Vet J 75:504–511
     [Google Scholar]
  10. Dybvig K., Voelker L.L. 1996; Molecular biology of myco-plasmas.. Annu Rev Microbiol 50:25–57
     [Google Scholar]
  11. Etheridge J.R., Cottew G.S., Lloyd LC. 1979; Isolation of Mycoplasma hyopneumoniae from lesions in experimentally infected pigs.. Aust Vet J 55:356–359
     [Google Scholar]
  12. Fagan P.K., Djordjevic S.P., Eamens G.J., Chin J., Walker M.J. 1996; Molecular characterisation of a ribonucleotide reductase (nrdF) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia.. Infect Immun 64:1014–1018
     [Google Scholar]
  13. Haldimann A., Nicolet J., Frey J. 1993; DNA sequence determination and biochemical analysis of the immunogenic protein P36 the lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae.. J Gen Microbiol 139:317–323
     [Google Scholar]
  14. Hsu T., Artiushin S., Minion F.C. 1997; Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.. J Bacterial 179:1317–1323
     [Google Scholar]
  15. King K.W., Faulds D.H., Rosey E.L., Yancey R.J. 1997; Characterization of the gene encoding Mhpl from Mycoplasma hyopneumoniae and examination of Mhpl’s vaccine potential.. Vaccine 15:25–35
     [Google Scholar]
  16. Klinkert M.-Q., Herrmann R., Schaller H. 1985; Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library.. Infect Immun 49:329–335
     [Google Scholar]
  17. Kyte J. 1995; Sequences of polymers.. In Structure in Protein Chemistry pp. 71–102 London & New York: Garland Publishing;
     [Google Scholar]
  18. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 227:680–685
     [Google Scholar]
  19. Leininger E., Roberts M., Kenimer J.G., Charles I.G., Fairweather N., Novotny P., Brennan M.J. 1991; Pertactin, an Arg-Gly-Asp-containing Bortedella pertussis surface protein that promotes adherence of mammalian cells.. Proc Natl Acad Sci USA 88345–349
     [Google Scholar]
  20. Petes T.D., Hill C.W. 1988; Recombination between repeated genes in microorganisms.. Annu Rev Genet 22:147–168
     [Google Scholar]
  21. Proft T., Helmut H., Layh-Schmitt G., Herrmann R. 1995; The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton-X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH.. J Bacteriol 177:3370–3378
     [Google Scholar]
  22. Razin S. 1979; Membrane proteins.. In The Mycoplasmas 1 Edited by Barile M. F., Razin S. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  23. Ro L-H., Chen R.-J., Shiuan D. 1994; Rapid purification of antiserum against Mycoplasma hyopneumoniae by an efficient absorption method.. J Biochem Biophys Methods 28:155–159
     [Google Scholar]
  24. Rosengarten R., Yogev D. 1996; Variant colony surface antigenic phenotypes within mycoplasma strain populations: implications for species identification and strain standardization.. J Clin Microbiol 34:149–158
     [Google Scholar]
  25. Ruoslahti E. 1996; RGD and other recognition sequences for integrins.. Annu Rev Cell Dev Biol 12:697–715
     [Google Scholar]
  26. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  27. Scarman A.L., Chin J.C., Eamens G.J., Delaney S.F., Djordjevic S.P. 1997; Identification of novel species-specific antigens of Mycoplasma hyopneumoniae by preparative SDS- PAGE ELISA profiling.. Microbiology 143:663–673
     [Google Scholar]
  28. Stemke G.W., Laiget F., Grau O., Bove J.M. 1992; Phylogenetic relationships of the three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis and complete 16S rRNA sequence of M. flocculare.. Int J Syst Bacteriol 42:220–225
     [Google Scholar]
  29. Stemke G.W., Phan R., Young T.F., Ross R.F. 1994; Differentiation of Mycoplasma hyopneumoniae, M. flocculare and M. hyorhinis on the basis of amplification of a 16S rRNA gene sequence.. Am J Vet Res 55:81–84
     [Google Scholar]
  30. Strasser M., Frey J., Bestetti G., Kobisch M., Nicolet J. 1991; Cloning and expression of a species-specific early immunogenic 36 kDa protein of Mycoplasma hyopneumoniae in E. coli.. Infect Immun 59:1217–1222
     [Google Scholar]
  31. Tajima M., Yagihashi T. 1982; Interaction of Mycoplasma hyopneumoniae with the porcine respiratory epithelium as observed by electron microscopy.. Infect Immun 37:1162–1169
     [Google Scholar]
  32. Wästfelt M., Stäihammar-Carlemalm M., Delisses A., Cabezon T., Lindahl G. 1996; Identification of a family of streptococcal surface proteins with extremely repetitive structure.. J Biol Chem 271:18892–18897
     [Google Scholar]
  33. Wieslander Å., Boyer M.J., Wróblewski H. 1992; Membrane protein structure.. In Mycoplasmas: Molecular Biology and Pathogenesis pp. 93–112 Edited by Maniloff J., McElhaney R. N., Finch L. R., Baseman J. B. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  34. Williamson M.P. 1994; The structure and function of prolinerich regions in proteins.. Biochem J 297:249–260
     [Google Scholar]
  35. Wren B.W. 1991; A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences.. Mol Microbiol 5:797–803
     [Google Scholar]
  36. Yamao F., Muto A., Kawauchi Y., Iwami M., Iwagami S., Azumi Y., Osawa S. 1985; UGA is read as tryptophan in Mycoplasma capricolum.. Proc Natl Acad Sci USA 822306–2309
     [Google Scholar]
  37. Yang Y., Gabriel D.W. 1995; Intragenic recombinant of a single plant pathogen gene provides a mechanism for the evolution of new host specificities.. J Bacteriol 177:4963–4968
     [Google Scholar]
  38. Young T.F., Chiang Y.-W., Ross R.F. 1990; Evaluation of local and systemic humoral immune responses to Mycoplasma hyopneumoniae.. Proceedings of the 11th Congress of the In-ternational Pig Veterinary Society p. 97
     [Google Scholar]
  39. Young T.F., Zhang Q., Ross R.F. 1992; Analysis of virulence- associated antigens of Mycoplasma hyopneumoniae.. Int Organ Mycoplasmol (IOM) Lett 2:321
     [Google Scholar]
  40. Zar J.H. 1984; Multiple comparisons.. In Biostatistical Analysis, 2nd edn. pp. 108–205 New Jersey: Prentice Hall Inc;
     [Google Scholar]
  41. Zhang Q., Wise K.S. 1996; Molecular basis of size and antigenic variation of a Mycoplasma hominis adhesin encoded by divergent vaa genes.. Infect Immun 64:2737–2744
     [Google Scholar]
  42. Zhang Q., Young T. F., Ross R. F. 1994a; Microtitre plate adherence assay and receptor analogues for Mycoplasma hyopneumoniae.. Infect Immun 62:1616–1622
     [Google Scholar]
  43. Zhang Q., Young T. F., Ross R. F. 1994b; Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells.. Infect Immun 62:4367–4373
     [Google Scholar]
  44. Zhang Q., Young T.F., Ross R.F. 1995; Identification and characterisation of a Mycoplasma hyopneumoniae adhesin.. Infect Immun 63:1013–1019
     [Google Scholar]
  45. Zheng X., Teng L.J., Watson H.L., Glass J.I., Blanchard A., Cassell G.H. 1995; Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with antigenic size variation.. Infect Immun 63:891–898
     [Google Scholar]
  46. Zielinski G.C., Ross R.F. 1990; Effect on growth in cell cultures and strain on virulence of Mycoplasma hyopneumoniae for swine.. Am J Vet Res 51:344–348
     [Google Scholar]
  47. Zielinski G.C., Young T.F., Ross R.F. 1993; Adherence of Mycoplasma hyopneumoniae to porcine ciliated respiratory tract cells.. Am J Vet Res 54:1262–1269
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-144-7-1931
Loading
Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae
Microbiology 144, 1931 (1998); https://doi.org/10.1099/00221287-144-7-1931
/content/journal/micro/10.1099/00221287-144-7-1931
/content/journal/micro/10.1099/00221287-144-7-1931
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error