1887

Abstract

is an opportunistic bacterial pathogen implicated in a variety of devastating conditions. Its flexibility as a pathogen is attributed to a myriad of virulence factors and regulatory elements that respond to prevailing environmental conditions. ExoS and ExoT are type III secreted effector proteins, regulated by the transcriptional activator ExsA, that can inhibit invasion of epithelial cells by cytotoxic strains of . This study sought to understand why invasive strains, which can secrete both ExoS and ExoT, still invade epithelial cells. The results showed that LasA and elastase (LasB), which are regulated by the Las and Rhl quorum-sensing systems, modulated invasion. Mutation of and/or reduced invasion, which was not fully restored by extracellularly added LasB, conditioned medium containing LasA and LasB, or EGTA pretreatment of cells. This indicated that protease effects on invasion involved factors additional to tight junction disruption and subsequent alterations to cell polarity. Upon mutation of and/or , steady-state levels of ExoS and ExoT were increased in culture medium of grown under conditions stimulatory for these toxins. The increase in ExoS was significantly correlated with reduced invasion. experiments showed that purified LasB degraded recombinant ExoS. Taken together, these studies suggest a mechanism by which invasive strains can synthesize inhibitors of invasion, ExoS and ExoT, yet still invade epithelial cells. By this mechanism, LasA and LasB decrease the levels of the toxins directly or indirectly, and thus reduce inhibition of invasion.

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2003-08-01
2019-08-17
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References

  1. Allewelt, M., Coleman, F. T., Grout, M., Priebe, G. P. & Pier, G. B. ( 2000; ). Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread. Infect Immun 68, 3998–4004.[CrossRef]
    [Google Scholar]
  2. Azghani, A. O. ( 1996; ). Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A. Am J Resp Cell Mol Biol 15, 132–140.[CrossRef]
    [Google Scholar]
  3. Braun, P., Tommassen, J. & Filloux, A. ( 1996; ). Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa. Mol Microbiol 19, 297–306.[CrossRef]
    [Google Scholar]
  4. Calfee, M. W., Coleman, J. P. & Pesci, E. C. ( 2001; ). Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa. Proc Natl Acad Sci 98, 11633–11637.[CrossRef]
    [Google Scholar]
  5. Carmeli, Y., Troillet, N., Karchmer, A. W. & Samore, M. J. ( 1999; ). Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa. Arch Intern Med 159, 127–132.
    [Google Scholar]
  6. Cheng, L. W. & Schneewind, O. ( 2000; ). Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol 8, 214–220.[CrossRef]
    [Google Scholar]
  7. Comolli, J. C., Hauser, A. R., Waite, L., Whitchurch, C. B., Mattick, J. S. & Engel, J. N. ( 1999; ). Pseudomonas aeruginosa gene products pilT and pilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect Immun 67, 3625–3630.
    [Google Scholar]
  8. Cornelis, G. R. & Van Gijsegem, F. ( 2000; ). Assembly and function of type III secretory systems. Annu Rev Microbiol 54, 735–774.[CrossRef]
    [Google Scholar]
  9. Cowan, C., Jones, H. A., Kaya, Y. H., Perry, R. D. & Straley, S. C. ( 2000; ). Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin. Infect Immun 68, 4523–4530.[CrossRef]
    [Google Scholar]
  10. Cowell, B. A., Wu, C. & Fleiszig, S. M. J. ( 1999; ). Use of an animal model in studies of bacterial corneal infection. Inst Lab Animal Res J 40, 43–50.[CrossRef]
    [Google Scholar]
  11. Cowell, B. A., Chen, D. Y., Frank, D. W., Vallis, A. J. & Fleiszig, S. M. J. ( 2000; ). ExoT of cytotoxic Pseudomonas aeruginosa prevents uptake by corneal epithelial cells. Infect Immun 68, 403–406.[CrossRef]
    [Google Scholar]
  12. Cowell, B. A., Weissman, B. A., Yeung, K. K., Johnson, L., Ho, S., Van, R., Bruckner, D., Mondino, B. & Fleiszig, S. M. J. ( 2003; ). Phenotype of Pseudomonas aeruginosa isolates causing corneal infection between 1997 and 2000. Cornea 22, 131–134.[CrossRef]
    [Google Scholar]
  13. Finck-Barbançon, V., Goranson, J., Zhu, L., Wiener-Kronish, J. P., Fleiszig, S. M. J., Wu, C., Mende-Mueller, L. & Frank, D. W. ( 1997; ). ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol Microbiol 25, 547–557.[CrossRef]
    [Google Scholar]
  14. Fleiszig, S. M. J., Zaidi, T. S. & Pier, G. B. ( 1995; ). Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro. Infect Immun 63, 4072–4077.
    [Google Scholar]
  15. 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.
    [Google Scholar]
  16. Fleiszig, S. M. J., Wiener-Kronish, J. P., Miyazaki, H., Vallas, V., Mostov, K. E., Kanada, D., Sawa, T., Yen, T. S. B. & Frank, D. W. ( 1997a; ). Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65, 579–586.
    [Google Scholar]
  17. Fleiszig, S. M. J., Evans, D. J., Do, N., Vallas, V., Shin, S. & Mostov, K. E. ( 1997b; ). Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity. Infect Immun 65, 2861–2867.
    [Google Scholar]
  18. Fleiszig, S. M. J., Arora, S. K., Van, R. & Ramphal, R. ( 2001; ). FlhA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa, plays a role in internalization by corneal epithelial cells. Infect Immun 69, 4931–4937.[CrossRef]
    [Google Scholar]
  19. Garrity-Ryan, L., Kazmierczak, B., Kowal, R., Comolli, J., Hauser, A. & Engel, J. N. ( 2000; ). The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages. Infect Immun 68, 7100–7113.[CrossRef]
    [Google Scholar]
  20. Gustin, J. K. ( 1998; ). Analysis of the Pseudomonas aeruginosa LasA protease. PhD Dissertation, University of Tennessee, Memphis, TN.
  21. Ha, U. & Jin, S. ( 2001; ). Growth phase-dependent invasion of Pseudomonas aeruginosa and its survival within HeLa cells. Infect Immun 69, 4398–4406.[CrossRef]
    [Google Scholar]
  22. Kadurugamuwa, J. L. & Beveridge, T. J. ( 1997; ). Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. J Antimicrob Chemother 40, 615–621.[CrossRef]
    [Google Scholar]
  23. Kamath, S., Kapatral, V. & Chakrabarty, A. M. ( 1998; ). Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis. Mol Microbiol 30, 933–941.[CrossRef]
    [Google Scholar]
  24. Kessler, E. & Safrin, M. ( 1994; ). The propeptide of Pseudomonas aeruginosa elastase acts as an elastase inhibitor. J Biol Chem 269, 22726–22731.
    [Google Scholar]
  25. Kessler, E., Safrin, M., Olson, J. C. & Ohman, D. E. ( 1993; ). Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease. J Biol Chem 268, 7503–7508.
    [Google Scholar]
  26. Kessler, E., Safrin, M., Abrams, W. R., Rosenbloom, J. & Ohman, D. E. ( 1997; ). Inhibitors and specificity of Pseudomonas aeruginosa LasA. J Biol Chem 272, 9884–9889.[CrossRef]
    [Google Scholar]
  27. Kessler, E., Safrin, M., Gustin, J. K. & Ohman, D. E. ( 1998; ). Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 273, 30225–30231.[CrossRef]
    [Google Scholar]
  28. Kudoh, I., Wiener-Kronish, J. P., Hashimoto, S., Pittet, J.-F. & Frank, D. (1994; ). Exoproduct secretions of Pseudomonas aeruginosa strains influence severity of alveolar epithelial injury. Am J Physiol 267, L551–556.
    [Google Scholar]
  29. Kulich, S. M., Frank, D. W. & Barbieri, J. T. ( 1993; ). Purification and characterization of recombinant exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun 61, 307–313.
    [Google Scholar]
  30. McCarthy, M. ( 2000; ). Pseudomonas genome reveals a formidable foe. Lancet 356, 918.[CrossRef]
    [Google Scholar]
  31. McIver, K. S., Kessler, E., Olson, J. C. & Ohman, D. E. ( 1995; ). The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18, 877–889.[CrossRef]
    [Google Scholar]
  32. O'Brien, T. & Hazlett, L. ( 1996; ). Pathogenesis of ocular microbial infection. In Ocular Infection and Immunity, pp. 200–214. Edited by J. Pepose, G. Holland & K. Wilhelmus. Baltimore, MD: Mosby Year Book.
  33. Pesci, E. C., Pearson, J. P., Seed, P. C. & Iglewski, B. H. ( 1997; ). Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 179, 3127–3132.
    [Google Scholar]
  34. Pier, G. B., Grout, M., Zaidi, T. S., Olsen, J. C., Johnson, L. G., Yankaskas, J. R. & Goldberg, J. B. ( 1996; ). Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271, 64–67.[CrossRef]
    [Google Scholar]
  35. Pillar, C. M., Hazlett, L. D. & Hobden, J. A. ( 2000; ). Alkaline protease-deficient mutants of Pseudomonas aeruginosa are virulent in the eye. Curr Eye Res 21, 730–739.[CrossRef]
    [Google Scholar]
  36. Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors ( 2000; ). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef]
    [Google Scholar]
  37. Tang, H. B., Dimango, E., Brian, M. J., Gambello, M. J., Iglewski, B. J., Goldberg, J. B. & Prince, A. ( 1996; ). Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect Immun 64, 37–43.
    [Google Scholar]
  38. Toder, D. S., Gambello, M. J. & Iglewski, B. H. ( 1991; ). Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR. Mol Microbiol 5, 2003–2010.[CrossRef]
    [Google Scholar]
  39. Twining, S. S. ( 1984; ). Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem 143, 30–34.[CrossRef]
    [Google Scholar]
  40. 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.
    [Google Scholar]
  41. Vogel, H. J. & Bonner, D. M. ( 1956; ). Acetylornithase of Escherichia coli: partial purification and some properties. J Biol Chem 218, 97–106.
    [Google Scholar]
  42. Yahr, T. L., Barbieri, J. T. & Frank, D. W. ( 1996; ). Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J Bact 178, 1412–1419.
    [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.
    [Google Scholar]
  44. Zaidi, T. S., Fleiszig, S. M. J., Preston, M. J., Goldberg, J. B. & Pier, G. B. ( 1996; ). Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa. Invest Ophthalmol Vis Sci 37, 976–986.
    [Google Scholar]
  45. Zhu, H., Thuruthyil, S. J., Kjelleberg, S., Rice, S., Givskov, M. & Willcox, M. D. P. ( 2001; ). Contribution of quorum-sensing systems to the virulence of Pseudomonas aeruginosa during corneal infections. Invest Ophthalmol Vis Sci 42, S514.
    [Google Scholar]
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