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Volume 155, Issue 2

Verification of a topology model of PorT as an integral outer-membrane protein in Free

Abstract

PorT is a membrane-associated protein shown to be essential for the maturation and secretion of a class of cysteine proteinases, the gingipains, from the periodontal pathogen . It was previously reported that PorT is located on the periplasmic surface of the inner membrane to function as a chaperone for the maturing proteinases. Our modelling suggested it to be an integral outer-membrane protein with eight anti-parallel, membrane-traversing -strands. In this report, the outer-membrane localization model was confirmed by the structural and functional tolerance of PorT to hexahistidine (6×His) tag insertions at selected locations within the protein using site-directed mutagenesis. Interestingly, those PorT mutations adversely affecting gingipain secretion enhanced expression of the gene but at the same time suppressed the transcription of the gingipain gene. Further, PorT mutants deficient in gingipain activities produced significantly more di- and triaminopeptidase activities. PorT homologues have been found in restricted members of the phylum where there is potential for PorT to participate in the maturation and secretion of proteins with characteristic C-terminal domains (CTDs). Knowledge of the cellular localization of PorT will enable analysis of the role of this protein in a new secretory pathway for the export of gingipains and other CTD-class proteins.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): AP, alkaline phosphatase , CTD, C-terminal domain , IM, inner membrane , OM, outer membrane and TLCK, tosyl-l-lysine chloromethyl ketone
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2009-02-01
2024-04-19
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References

  1. Banbula A., Mak P., Bugno M., Silberring J., Dubin A., Nelson D., Travis J., Potempa J. 1999; Prolyl tripeptidyl peptidase from Porphyromonas gingivalis . A novel enzyme with possible pathological implications for the development of periodontitis. J Biol Chem 274:9246–9252
     [Google Scholar]
  2. Barnard T. J., Dautin N., Lukacik P., Bernstein H. D., Buchanan S. K. 2007; Autotransporter structure reveals intra-barrel cleavage followed by conformational changes. Nat Struct Mol Biol 14:1214–1220
     [Google Scholar]
  3. Bigelow H. R., Petrey D. S., Liu J., Przybylski D., Rost B. 2004; Predicting transmembrane beta-barrels in proteomes. Nucleic Acids Res 32:2566–2577
     [Google Scholar]
  4. Chiu J., March P. E., Lee R., Tillett D. 2004; Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100 % efficiency in 4 h. Nucleic Acids Res 32:e174
     [Google Scholar]
  5. Cianciotto N. P. 2005; Type II secretion: a protein secretion system for all seasons. Trends Microbiol 13:581–588
     [Google Scholar]
  6. Eleaume H., Jabbouri S. 2004; Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth. J Microbiol Methods 59:363–370
     [Google Scholar]
  7. Filip C., Fletcher G., Wulff J. L., Earhart C. F. 1973; Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115:717–722
     [Google Scholar]
  8. Freudl R. 1989; Insertion of peptides into cell-surface-exposed areas of the Escherichia coli OmpA protein does not interfere with export and membrane assembly. Gene 82:229–236
     [Google Scholar]
  9. Freudl R., Schwarz H., Stierhof Y. D., Gamon K., Hindennach I., Henning U. 1986; An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export. J Biol Chem 261:11355–11361
     [Google Scholar]
  10. Galdiero S., Galdiero M., Pedone C. 2007; β -Barrel membrane bacterial proteins: structure, function, assembly and interaction with lipids. Curr Protein Pept Sci 8:63–82
     [Google Scholar]
  11. Gerlach R. G., Hensel M. 2007; Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens. Int J Med Microbiol 297:401–415
     [Google Scholar]
  12. Ghosh P. 2004; Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68:771–795
     [Google Scholar]
  13. Henderson I. R., Navarro-Garcia F., Desvaux M., Fernandez R. C., Ala'Aldeen D. 2004; Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68:692–744
     [Google Scholar]
  14. Imamura T. 2003; The role of gingipains in the pathogenesis of periodontal disease. J Periodontol 74:111–118
     [Google Scholar]
  15. Imamura T., Travis J., Potempa J. 2003; The biphasic virulence activities of gingipains: activation and inactivation of host proteins. Curr Protein Pept Sci 4:443–450
     [Google Scholar]
  16. Jones D. T. 1999; Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202
     [Google Scholar]
  17. Kamaguchi A., Nakayama K., Ohyama T., Watanabe T., Okamoto M., Baba H. 2001; Coaggregation of Porphyromonas gingivalis and Prevotella intermedia . Microbiol Immunol 45:649–656
     [Google Scholar]
  18. Koebnik R., Locher K. P., Van Gelder P. 2000; Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol Microbiol 37:239–253
     [Google Scholar]
  19. Laskowski R. A., Watson J. D., Thornton J. M. 2005a; ProFunc: a server for predicting protein function from 3D structure. Nucleic Acids Res 33:W89–W93
     [Google Scholar]
  20. Laskowski R. A., Watson J. D., Thornton J. M. 2005b; Protein function prediction using local 3D templates. J Mol Biol 351:614–626
     [Google Scholar]
  21. Mougous J. D., Cuff M. E., Raunser S., Shen A., Zhou M., Gifford C. A., Goodman A. L., Joachimiak G., Ordoñez C. L. other authors 2006; A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312:1526–1530
     [Google Scholar]
  22. Nakamura S., Takeuchi A., Masamoto Y., Abiko Y., Hayakawa M., Takiguchi H. 1992; Cloning of the gene encoding a glycylprolyl aminopeptidase from Porphyromonas gingivalis . Arch Oral Biol 37:807–812
     [Google Scholar]
  23. Nelson K. E., Fleischmann R. D., DeBoy R. T., Paulsen I. T., Fouts D. E., Eisen J. A., Daugherty S. C., Dodson R. J., Durkin A. S. other authors 2003; Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis strain W83. J Bacteriol 185:5591–5601
     [Google Scholar]
  24. Nguyen K. A., Travis J., Potempa J. 2007; Does the importance of the C-terminal residues in the maturation of RgpB from Porphyromonas gingivalis reveal a novel mechanism for protein export in a subgroup of Gram-negative bacteria?. J Bacteriol 189:833–843
     [Google Scholar]
  25. Nikaido H. 1994; Isolation of outer membranes. Methods Enzymol 235:225–234
     [Google Scholar]
  26. Nikolich M. P., Shoemaker N. B., Salyers A. A. 1992; A Bacteroides tetracycline resistance gene represents a new class of ribosome protection tetracycline resistance. Antimicrob Agents Chemother 36:1005–1012
     [Google Scholar]
  27. Nollmann M., Crisona N. J., Arimondo P. B. 2007; Thirty years of Escherichia coli DNA gyrase: from in vivo function to single-molecule mechanism. Biochimie 89:490–499
     [Google Scholar]
  28. O'Brien-Simpson N. M., Paolini R. A., Hoffmann B., Slakeski N., Dashper S. G., Reynolds E. C. 2001; Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model. Infect Immun 69:7527–7534
     [Google Scholar]
  29. Okamoto K., Nakayama K., Kadowaki T., Abe N., Ratnayake D. B., Yamamoto K. 1998; Involvement of a lysine-specific cysteine proteinase in hemoglobin adsorption and heme accumulation by Porphyromonas gingivalis . J Biol Chem 273:21225–21231
     [Google Scholar]
  30. Potempa J., Pike R., Travis J. 1995; The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain. Infect Immun 63:1176–1182
     [Google Scholar]
  31. Potempa J., Sroka A., Imamura T., Travis J. 2003; Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis : structure, function and assembly of multidomain protein complexes. Curr Protein Pept Sci 4:397–407
     [Google Scholar]
  32. Pukatzki S., Ma A. T., Sturtevant D., Krastins B., Sarracino D., Nelson W. C., Heidelberg J. F., Mekalanos J. J. 2006; Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 103:1528–1533
     [Google Scholar]
  33. Robert V., Volokhina E. B., Senf F., Bos M. P., Van Gelder P., Tommassen J. 2006; Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol 4:e377
     [Google Scholar]
  34. Saiki K., Konishi K. 2007; Identification of a Porphyromonas gingivalis novel protein Sov required for the secretion of gingipains. Microbiol Immunol 51:483–491
     [Google Scholar]
  35. Sali A., Blundell T. L. 1993; Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815
     [Google Scholar]
  36. Sato K., Sakai E., Veith P. D., Shoji M., Kikuchi Y., Yukitake H., Ohara N., Naito M., Okamoto K. other authors 2005; Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis . J Biol Chem 280:8668–8677
     [Google Scholar]
  37. Schmidt T. G., Skerra A. 2007; The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535
     [Google Scholar]
  38. Schnaitman C. A. 1971; Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol 108:545–552
     [Google Scholar]
  39. Schulz G. E. 2002; The structure of bacterial outer membrane proteins. Biochim Biophys Acta 1565:308–317
     [Google Scholar]
  40. Seers C. A., Slakeski N., Veith P. D., Nikolof T., Chen Y. Y., Dashper S. G., Reynolds E. C. 2006; The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal-domain family found in Porphyromonas gingivalis . J Bacteriol 188:6376–6386
     [Google Scholar]
  41. Söding J., Biegert A., Lupas A. N. 2005; The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33:W244–W248
     [Google Scholar]
  42. Voulhoux R., Bos M. P., Geurtsen J., Mols M., Tommassen J. 2003; Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299:262–265
     [Google Scholar]
  43. Weinberg A., Belton C. M., Park Y., Lamont R. J. 1997; Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 65:313–316
     [Google Scholar]
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Verification of a topology model of PorT as an integral outer-membrane protein in Porphyromonas gingivalis
Microbiology 155, 328 (2009); https://doi.org/10.1099/mic.0.024323-0
/content/journal/micro/10.1099/mic.0.024323-0
/content/journal/micro/10.1099/mic.0.024323-0
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