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Volume 61, Issue 2

Characterization of protease IV expression in clinical isolates Free

Abstract

Expression of protease IV by during ocular infections contributes significantly to tissue damage. However, several strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of were determined and compared to strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorum-sensing gene () mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5–99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAOΔ mutants and the two clinical isolates with a gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system.

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© 2012 SGM
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2012-02-01
2024-04-18
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References

  1. Arevalo-Ferro C., Hentzer M., Reil G., Görg A., Kjelleberg S., Givskov M., Riedel K., Eberl L. 2003; Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics. Environ Microbiol 5:1350–1369 [View Article][PubMed]
     [Google Scholar]
  2. Battle S. E., Rello J., Hauser A. R. 2009; Genomic islands of Pseudomonas aeruginosa. FEMS Microbiol Lett 290:70–78 [View Article][PubMed]
     [Google Scholar]
  3. Beatson S. A., Whitchurch C. B., Semmler A. B., Mattick J. S. 2002; Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa. J Bacteriol 184:3598–3604 [View Article][PubMed]
     [Google Scholar]
  4. Bleves S., Soscia C., Nogueira-Orlandi P., Lazdunski A., Filloux A. 2005; Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1. J Bacteriol 187:3898–3902 [View Article][PubMed]
     [Google Scholar]
  5. Caballero A. R., Moreau J. M., Engel L. S., Marquart M. E., Hill J. M., O’Callaghan R. J. 2001; Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases. Anal Biochem 290:330–337 [View Article][PubMed]
     [Google Scholar]
  6. Caballero A., Thibodeaux B., Marquart M., Traidej M., O’Callaghan R. 2004; Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases. Invest Ophthalmol Vis Sci 45:522–530 [View Article][PubMed]
     [Google Scholar]
  7. Chapon-Hervé V., Akrim M., Latifi A., Williams P., Lazdunski A., Bally M. 1997; Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa. Mol Microbiol 24:1169–1178 [View Article][PubMed]
     [Google Scholar]
  8. Chun C. K., Ozer E. A., Welsh M. J., Zabner J., Greenberg E. P. 2004; Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia. Proc Natl Acad Sci U S A 101:3587–3590 [View Article][PubMed]
     [Google Scholar]
  9. Engel L. S., Hobden J. A., Moreau J. M., Callegan M. C., Hill J. M., O’Callaghan R. J. 1997; Pseudomonas deficient in protease IV has significantly reduced corneal virulence. Invest Ophthalmol Vis Sci 38:1535–1542[PubMed]
     [Google Scholar]
  10. Engel L. S., Hill J. M., Caballero A. R., Green L. C., O’Callaghan R. J. 1998; Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa. J Biol Chem 273:16792–16797 [View Article][PubMed]
     [Google Scholar]
  11. Fleiszig S. M. J., Zaidi T. S., Preston M. J., Grout M., Evans D. J., Pier G. B. 1996; Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa. Infect Immun 64:2288–2294[PubMed]
     [Google Scholar]
  12. Fleiszig S. M. J., Wiener-Kronish J. P., Miyazaki H., Vallas V., Mostov K. E., Kanada D., Sawa T., Yen T. S., Frank D. W. 1997; Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65:579–586[PubMed]
     [Google Scholar]
  13. Gambello M. J., Kaye S., Iglewski B. H. 1993; LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infect Immun 61:1180–1184[PubMed]
     [Google Scholar]
  14. Hall T. A. 1999; BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
     [Google Scholar]
  15. Hazlett L. D. 2002; Pathogenic mechanisms of P. aeruginosa keratitis: a review of the role of T cells, Langerhans cells, PMN, and cytokines. DNA Cell Biol 21:383–390 [View Article][PubMed]
     [Google Scholar]
  16. Holloway B. W. 1965; Variations in restriction and modification of bacteriophage following Increase of growth temperature of Pseudomonas aeruginosa. Virology 25:634–642 [View Article][PubMed]
     [Google Scholar]
  17. Holloway B. W., Krishnapillai V., Morgan A. F. 1979; Chromosomal genetics of Pseudomonas. Microbiol Rev 43:73–102[PubMed]
     [Google Scholar]
  18. Kessler E., Blumberg S. 1987; Specific inhibitors of Pseudomonas aeruginosa elastase as potential drugs for the treatment of Pseudomonas keratitis. Antibiot Chemother 39:102–112[PubMed]
     [Google Scholar]
  19. Kim E. J., Wang W., Deckwer W. D., Zeng A. P. 2005; Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density. Microbiology 151:1127–1138 [View Article][PubMed]
     [Google Scholar]
  20. Lamont I. L., Beare P. A., Ochsner U., Vasil A. I., Vasil M. L. 2002; Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 99:7072–7077 [View Article][PubMed]
     [Google Scholar]
  21. Latifi A., Foglino M., Tanaka K., Williams P., Lazdunski A. 1996; A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21:1137–1146 [View Article][PubMed]
     [Google Scholar]
  22. Leber T. M., Balkwill F. R. 1997; Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem 249:24–28 [View Article][PubMed]
     [Google Scholar]
  23. Lee D. G., Urbach J. M., Wu G., Liberati N. T., Feinbaum R. L., Miyata S., Diggins L. T., He J. X., Saucier M. other authors 2006; Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol 7:R90 [View Article][PubMed]
     [Google Scholar]
  24. Marquart M. E., Caballero A. R., Chomnawang M., Thibodeaux B. A., Twining S. S., O’Callaghan R. J. 2005; Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions. Invest Ophthalmol Vis Sci 46:3761–3768 [View Article][PubMed]
     [Google Scholar]
  25. Mercer A. A., Loutit J. S. 1979; Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions. J Bacteriol 140:37–42[PubMed]
     [Google Scholar]
  26. O’Callaghan R. J., Engel L. S., Hobden J. A., Callegan M. C., Green L. C., Hill J. M. 1996; Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence. Invest Ophthalmol Vis Sci 37:534–543[PubMed]
     [Google Scholar]
  27. Ochsner U. A., Vasil A. I., Vasil M. L. 1995; Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol 177:7194–7201[PubMed]
     [Google Scholar]
  28. Olsen G. J., Matsuda H., Hagstrom R., Overbeek R. 1994; fastDNAmL: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci 10:41–48[PubMed]
     [Google Scholar]
  29. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. 1993; Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260:1127–1130 [View Article][PubMed]
     [Google Scholar]
  30. Pearson J. P., Gray K. M., Passador L., Tucker K. D., Eberhard A., Iglewski B. H., Greenberg E. P. 1994; Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A 91:197–201 [View Article][PubMed]
     [Google Scholar]
  31. Pearson J. P., Pesci E. C., Iglewski B. H. 1997; Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179:5756–5767[PubMed]
     [Google Scholar]
  32. Sandkvist M. 2001; Biology of type II secretion. Mol Microbiol 40:271–283 [View Article][PubMed]
     [Google Scholar]
  33. Sankaridurg P. R., Sharma S., Willcox M., Naduvilath T. J., Sweeney D. F., Holden B. A., Rao G. N. 2000; Bacterial colonization of disposable soft contact lenses is greater during corneal infiltrative events than during asymptomatic extended lens wear. J Clin Microbiol 38:4420–4424[PubMed]
     [Google Scholar]
  34. Singh G., Wu B., Baek M. S., Camargo A., Nguyen A., Slusher N. A., Srinivasan R., Wiener-Kronish J. P., Lynch S. V. 2010; Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on Pseudomonas quinolone signal concentration. Microb Pathog 49:196–203 [View Article][PubMed]
     [Google Scholar]
  35. Stewart R. M. K., Wiehlmann L., Ashelford K. E., Preston S. J., Frimmersdorf E., Campbell B. J., Neal T. J., Hall N., Tuft S. other authors 2011; Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections. J Clin Microbiol 49:993–1003 [View Article][PubMed]
     [Google Scholar]
  36. Stintzi A., Evans K., Meyer J. M., Poole K. 1998; Quorum-sensing and siderophore biosynthesis in Pseudomonas aeruginosa: lasR/lasI mutants exhibit reduced pyoverdine biosynthesis. FEMS Microbiol Lett 166:341–345 [View Article][PubMed]
     [Google Scholar]
  37. Stover C. K., Pham X. Q., Erwin A. L., Mizoguchi S. D., Warrener P., Hickey M. J., Brinkman F. S. L., Hufnagle W. O., Kowalik D. J. other authors 2000; Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964 [View Article][PubMed]
     [Google Scholar]
  38. Traidej M., Caballero A. R., Marquart M. E., Thibodeaux B. A., O’Callaghan R. J. 2003; Molecular analysis of Pseudomonas aeruginosa protease IV expressed in Pseudomonas putida. Invest Ophthalmol Vis Sci 44:190–196 [View Article][PubMed]
     [Google Scholar]
  39. Twining S. S., Kirschner S. E., Mahnke L. A., Frank D. W. 1993; Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest Ophthalmol Vis Sci 34:2699–2712[PubMed]
     [Google Scholar]
  40. Watson A. A., Alm R. A., Mattick J. S. 1996; Construction of improved vectors for protein production in Pseudomonas aeruginosa. Gene 172:163–164 [View Article][PubMed]
     [Google Scholar]
  41. Wilderman P. J., Vasil A. I., Johnson Z., Wilson M. J., Cunliffe H. E., Lamont I. L., Vasil M. L. 2001; Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa. Infect Immun 69:5385–5394 [View Article][PubMed]
     [Google Scholar]
  42. Winstanley C., Kaye S. B., Neal T. J., Chilton H. J., Miksch S., Hart C. A.Microbiology Ophthalmic Group 2005; Genotypic and phenotypic characteristics of Pseudomonas aeruginosa isolates associated with ulcerative keratitis. J Med Microbiol 54:519–526 [View Article][PubMed]
     [Google Scholar]
  43. Yahr T. L., Mende-Mueller L. M., Friese M. B., Frank D. W. 1997; Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol 179:7165–7168[PubMed]
     [Google Scholar]
  44. Zhu H., Thuruthyil S. J., Willcox M. D. P. 2002; Determination of quorum-sensing signal molecules and virulence factors of Pseudomonas aeruginosa isolates from contact lens-induced microbial keratitis. J Med Microbiol 51:1063–1070[PubMed]
     [Google Scholar]
  45. Zhu H., Bandara R., Conibear T. C. R., Thuruthyil S. J., Rice S. A., Kjelleberg S., Givskov M., Willcox M. D. P. 2004; Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection. Invest Ophthalmol Vis Sci 45:1897–1903 [View Article][PubMed]
     [Google Scholar]
  46. Zhu H., Conibear T. C. R., Bandara R., Aliwarga Y., Stapleton F., Willcox M. D. P. 2006; Type III secretion system-associated toxins, proteases, serotypes, and antibiotic resistance of Pseudomonas aeruginosa isolates associated with keratitis. Curr Eye Res 31:297–306 [View Article][PubMed]
     [Google Scholar]
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Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates
J. Med. Microbiol. 61, 180 (2012); https://doi.org/10.1099/jmm.0.034561-0
/content/journal/jmm/10.1099/jmm.0.034561-0
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