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Volume 146, Issue 10

mediated apoptosis requires the ADP-ribosylating activity of ExoS Free

Abstract

is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Using a tissue culture system, invasive strains of were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines. This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity. Induction of the type III secretion machinery, but not invasion of is required for induction of apoptosis. The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection. Mutants in fail to induce apoptosis and complementation with wild-type restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule. Analysis of activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.

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Keyword(s): ADP-ribosylation , apoptosis , ExoS , FCS, foetal calf serum , p.i., post infection , PI, propidium iodide , TNFα, tumour necrosis factor α and type III secretion
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2000-10-01
2025-04-24
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References

  1. Apodaca G., Bomsel M., Lindstedt R., Engel J., Frank D., Mostov K. E., Wiener-Kronish J. 1995; Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing. Infect Immun 63:1541–1551
     [Google Scholar]
  2. Buommino E., Morelli F., Metafora S., Rossano F., Perfetto B., Baroni A., Tufano M. A. 1999; Porin from Pseudomonas aeruginosa induces apoptosis in an epithelial cell line derived from rat seminal vesicles. Infect Immun 67:4794–4800
     [Google Scholar]
  3. Coburn J., Dillon S. T., Iglewski B. H., Gill D. M. 1989a; Exoenzyme S of Pseudomonas aeruginosa ADP-ribosylates the intermediate filament protein vimentin. Infect Immun 57:996–998
     [Google Scholar]
  4. Coburn J., Wyatt R. T., Iglewski B. H., Gill D. M. 1989b; Several GTP-binding proteins, including p21c-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzyme S. J Biol Chem 264:9004–9008
     [Google Scholar]
  5. Cowell B. A., Chen D. Y., Frank D. W., Vallis A. J., Fleiszig S. M. 2000; ExoT of cytotoxic Pseudomonas aeruginosa prevents uptake by corneal epithelial cells. Infect Immun 68:403–406 [CrossRef]
     [Google Scholar]
  6. Evans D. J., Frank D. W., Finck-Barbancon V., Wu C., Fleiszig S. M. 1998; Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity. Infect Immun 66:1453–1459
     [Google Scholar]
  7. Finck-Barbancon V., Goranson J., Zhu L., Sawa T., Wiener-Kronish J. P., Fleiszig S. M., 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]
  8. Fleiszig S. M., 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
     [Google Scholar]
  9. Fraker P. J., King L. E., Lill-Elghanian D., Telford W. G. 1995; Quantification of apoptotic events in pure and heterogeneous populations of cells using the flow cytometer. Methods Cell Biol 46:57–76
     [Google Scholar]
  10. Frank D. W. 1997; The exoenzyme S regulon of Pseudomonas aeruginosa. Mol Microbiol 26:621–629 [CrossRef]
     [Google Scholar]
  11. Frithz-Lindsten E., Du Y., Rosqvist R., Forsberg A. 1997; Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments. Mol Microbiol 25:1125–1139 [CrossRef]
     [Google Scholar]
  12. Ganesan A. K., Frank D. W., Misra R. P., Schmidt G., Barbieri J. T. 1998; Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites. J Biol Chem 273:7332–7337 [CrossRef]
     [Google Scholar]
  13. Ganesan A. K., Vincent T. S., Olson J. C., Barbieri J. T. 1999; Pseudomonas aeruginosa exoenzyme S disrupts Ras-mediated signal transduction by inhibiting guanine nucleotide exchange factor-catalyzed nucleotide exchange. J Biol Chem 274:21823–21829 [CrossRef]
     [Google Scholar]
  14. Gherardini F. C., Hobbs M. M., Stamm L. V., Bassford P. J. Jr 1990; Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum. J Bacteriol 172:2996–3002
     [Google Scholar]
  15. Hafkemeyer P., Brinkmann U., Gottesman M. M., Pastan I. 1999; Apoptosis induced by Pseudomonas exotoxin: a sensitive and rapid marker for gene delivery in vivo. Hum Gene Ther 10:923–934 [CrossRef]
     [Google Scholar]
  16. Hahn H. P. 1997; The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review. Gene 192:99–108 [CrossRef]
     [Google Scholar]
  17. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
     [Google Scholar]
  18. Hauser A. R., Engel J. N. 1999; Pseudomonas aeruginosa induces type-III-secretion-mediated apoptosis of macrophages and epithelial cells. Infect Immun 67:5530–5537
     [Google Scholar]
  19. Hauser A. R., Kang P. J., Engel J. N. 1998; PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence. Mol Microbiol 27:807–818 [CrossRef]
     [Google Scholar]
  20. Hersh D., Monack D. M., Smith M. R., Ghori N., Falkow S., Zychlinsky A. 1999; The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc Natl Acad Sci U S A 96:2396–2401 [CrossRef]
     [Google Scholar]
  21. Hilbi H., Moss J. E., Hersh D.7 other authors 1999; Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J Biol Chem 273:32895–32900
     [Google Scholar]
  22. Holloway B. W., Krishnapillai V., Morgan A. F. 1979; Chromosomal genetics of Pseudomonas. Microbiol Rev 43:73–102
     [Google Scholar]
  23. Hovey A. K., Frank D. W. 1995; Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol 177:4427–4436
     [Google Scholar]
  24. Iglewski B. H., Kabat D. 1975; NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc Natl Acad Sci U S A 72:2284–2288 [CrossRef]
     [Google Scholar]
  25. Iglewski B. H., Sadoff J., Bjorn M. J., Maxwell E. S. 1978; Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A 75:3211–3215 [CrossRef]
     [Google Scholar]
  26. Knight D. A., Barbieri J. T. 1997; Ecto-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exoenzyme S. Infect Immun 65:3304–3309
     [Google Scholar]
  27. Kothakota S., Azuma T., Reinhard C.8 other authors 1997; Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278:294–298 [CrossRef]
     [Google Scholar]
  28. Kulich S. M., Frank D. W., Barbieri J. T. 1995; Expression of recombinant exoenzyme S of Pseudomonas aeruginosa. Infect Immun 63:1–8
     [Google Scholar]
  29. Liu P. V. 1966; The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. 3. Identity of the lethal toxins produced in vitro and in vivo. J Infect Dis 116:481–489 [CrossRef]
     [Google Scholar]
  30. Liu S., Kulich S. M., Barbieri J. T. 1996; Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S. Biochemistry 35:2754–2758 [CrossRef]
     [Google Scholar]
  31. McGuffie E. M., Frank D. W., Vincent T. S., Olson J. C. 1998; Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S. Infect Immun 66:2607–2613
     [Google Scholar]
  32. McGuffie E. M., Fraylick J. E., Hazen-Martin D. J., Vincent T. S., Olson J. C. 1999; Differential sensitivity of human epithelial cells to Pseudomonas aeruginosa exoenzyme S. Infect Immun 67:3494–3503
     [Google Scholar]
  33. McNeill H., Downward J. 1999; Apoptosis: Ras to the rescue in the fly eye. Curr Biol 9:R176–R179 [CrossRef]
     [Google Scholar]
  34. Morimoto H., Bonavida B. 1992; Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis: ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumour necrosis factor-alpha. J Immunol 149:2089–2094
     [Google Scholar]
  35. Nunn D., Bergman S., Lory S. 1990; Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 172:2911–2919
     [Google Scholar]
  36. Ohman D. E., Burns R. P., Iglewski B. H. 1980; Corneal infections in mice with toxin A and elastase mutants of Pseudomonas aeruginosa. J Infect Dis 142:547–555 [CrossRef]
     [Google Scholar]
  37. Olson J. C., Fraylick J. E., McGuffie E. M., Dolan K. M., Yahr T. L., Frank D. W., Vincent T. S. 1999; Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S. Infect Immun 67:2847–2854
     [Google Scholar]
  38. Orth K., Palmer L. E., Bao Z. Q., Stewart S., Rudolph A. E., Bliska J. B., Dixon J. E. 1999; Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia effector. Science 285:1920–1923 [CrossRef]
     [Google Scholar]
  39. Pederson K. J., Vallis A. J., Aktories K., Frank D. W., Barbieri J. T. 1999; The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. Mol Microbiol 32:393–401 [CrossRef]
     [Google Scholar]
  40. Salyers A. A., Whitt D. D. 1994 Bacterial Pathogenesis: a Molecular Approach Washington, DC: American Society for Microbiology;
     [Google Scholar]
  41. Simpson D. A., Ramphal R., Lory S. 1995; Characterization of Pseudomonas aeruginosa fliO, a gene involved in flagellar biosynthesis and adherence. Infect Immun 63:2950–2957
     [Google Scholar]
  42. Totten P. A., Lory S. 1990; Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol 172:7188–7199
     [Google Scholar]
  43. Tummler B., Kiewitz C. 1999; Cystic fibrosis: an inherited susceptibility to bacterial respiratory infections. Mol Med Today 5:351–358 [CrossRef]
     [Google Scholar]
  44. Vallis A. J., Finck-Barbancon V., Yahr T. L., Frank D. W. 1999; Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells. Infect Immun 67:2040–2044
     [Google Scholar]
  45. Verschueren H., van der Taelen I., Dewit J., De Braekeleer J., De Baetselier P., Aktories K., Just I. 1995; Effects of Clostridium botulinum C2 toxin and cytochalasin D on in vitro invasiveness, motility and F-actin content of a murine T-lymphoma cell line. Eur J Cell Biol 66:335–341
     [Google Scholar]
  46. Vincent T. S., Fraylick J. E., McGuffie E. M., Olson J. C. 1999; ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo. Mol Microbiol 32:1054–1064 [CrossRef]
     [Google Scholar]
  47. Wall D., Kaiser D. 1999; Type IV pili and cell motility. Mol Microbiol 32:1–10 [CrossRef]
     [Google Scholar]
  48. Yahr T. L., Goranson J., Frank D. W. 1996a; Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol Microbiol 22:991–1003 [CrossRef]
     [Google Scholar]
  49. Yahr T. L., Barbieri J. T., Frank D. W. 1996b; Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J Bacteriol 178:1412–1419
     [Google Scholar]
  50. 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]
  51. Yahr T. L., Vallis A. J., Hancock M. K., Barbieri J. T., Frank D. W. 1998; ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc Natl Acad Sci U S A 95:13899–13904 [CrossRef]
     [Google Scholar]
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Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS
Microbiology 146, 2531 (2000); https://doi.org/10.1099/00221287-146-10-2531
/content/journal/micro/10.1099/00221287-146-10-2531
/content/journal/micro/10.1099/00221287-146-10-2531
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