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. 1997 Jan;65(1):248–256. doi: 10.1128/iai.65.1.248-256.1997

Effects of differential expression of the 49-kilodalton exoenzyme S by Pseudomonas aeruginosa on cultured eukaryotic cells.

J C Olson 1, E M McGuffie 1, D W Frank 1
PMCID: PMC174583  PMID: 8975919

Abstract

Production of the ADP-ribosylating enzyme exoenzyme S (ExoS) by Pseudomonas aeruginosa has been associated with increased virulence. Previous studies, however, have been unable to confirm an effect of soluble ExoS in cell culture or animal model systems. To determine if bacteria must come in contact with target cells in order for an effect of ExoS to be observed, coculture systems were developed to compare the effects of ExoS- and non-ExoS-producing bacteria on eukaryotic cell function. The two P. aeruginosa strains used in these studies, 388 and 388delta exoS, maintained genetic identity, with the exception that strain 388delta exoS lacked production of the 49-kDa form of ExoS. When bacteria were cocultured with Detroit 532 fibroblastic cells, ExoS-producing 388 bacteria caused a significant decrease in DNA synthesis and viability compared to the decrease caused by non-ExoS-producing 388delta exoS bacteria. Maximal differences between the two strains were observed when 10(4) to 10(7) CFU of bacteria/ml were cocultured with Detroit cells for 4 or 6 h. Both strains were effective in eliminating Detroit cell DNA synthesis after a 20-h coculture period. Secreted ExoS had no effect on Detroit cell growth and viability, indicating that bacteria must have contact with target cells for the effect of ExoS on cellular function to be observed. Similar effects on cell proliferation and viability were observed when the two strains were cocultured with the KB epithelioid cell line. ExoS-associated decreases in eukaryotic cell viability were not found to be mediated by an inhibition of protein synthesis. These studies confirm that the 49-kDa ExoS contributes to the cellular pathogenesis of P. aeruginosa by interfering with eukaryotic cell growth and viability. In addition, the coculture system developed which recognizes this effect should provide a means for defining the function of ExoS in vivo.

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Selected References

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  1. Allaoui A., Scheen R., Lambert de Rouvroit C., Cornelis G. R. VirG, a Yersinia enterocolitica lipoprotein involved in Ca2+ dependency, is related to exsB of Pseudomonas aeruginosa. J Bacteriol. 1995 Aug;177(15):4230–4237. doi: 10.1128/jb.177.15.4230-4237.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Apodaca G., Bomsel M., Lindstedt R., Engel J., Frank D., Mostov K. E., Wiener-Kronish J. Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing. Infect Immun. 1995 Apr;63(4):1541–1551. doi: 10.1128/iai.63.4.1541-1551.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bjorn M. J., Pavlovskis O. R., Thompson M. R., Iglewski B. H. Production of exoenzyme S during Pseudomonas aeruginosa infections of burned mice. Infect Immun. 1979 Jun;24(3):837–842. doi: 10.1128/iai.24.3.837-842.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brumlik M. J., Storey D. G. Zinc and iron regulate translation of the gene encoding Pseudomonas aeruginosa elastase. Mol Microbiol. 1992 Feb;6(3):337–344. doi: 10.1111/j.1365-2958.1992.tb01476.x. [DOI] [PubMed] [Google Scholar]
  5. Carroll S. F., Collier R. J. Diphtheria toxin: quantification and assay. Methods Enzymol. 1988;165:218–225. doi: 10.1016/s0076-6879(88)65034-8. [DOI] [PubMed] [Google Scholar]
  6. Coburn J., Dillon S. T., Iglewski B. H., Gill D. M. Exoenzyme S of Pseudomonas aeruginosa ADP-ribosylates the intermediate filament protein vimentin. Infect Immun. 1989 Mar;57(3):996–998. doi: 10.1128/iai.57.3.996-998.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coburn J., Gill D. M. ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1991 Nov;59(11):4259–4262. doi: 10.1128/iai.59.11.4259-4262.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coburn J., Kane A. V., Feig L., Gill D. M. Pseudomonas aeruginosa exoenzyme S requires a eukaryotic protein for ADP-ribosyltransferase activity. J Biol Chem. 1991 Apr 5;266(10):6438–6446. [PubMed] [Google Scholar]
  9. Coburn J. Pseudomonas aeruginosa exoenzyme S. Curr Top Microbiol Immunol. 1992;175:133–143. doi: 10.1007/978-3-642-76966-5_7. [DOI] [PubMed] [Google Scholar]
  10. Coburn J., Wyatt R. T., Iglewski B. H., Gill D. M. Several GTP-binding proteins, including p21c-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzyme S. J Biol Chem. 1989 May 25;264(15):9004–9008. [PubMed] [Google Scholar]
  11. Frank D. W., Iglewski B. H. Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1991 Oct;173(20):6460–6468. doi: 10.1128/jb.173.20.6460-6468.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fu H., Coburn J., Collier R. J. The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2320–2324. doi: 10.1073/pnas.90.6.2320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goranson J., Frank D. W. Genetic analysis of exoenzyme S expression by Pseudomonas aeruginosa. FEMS Microbiol Lett. 1996 Jan 15;135(2-3):149–155. doi: 10.1111/j.1574-6968.1996.tb07981.x. [DOI] [PubMed] [Google Scholar]
  14. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iglewski B. H., Sadoff J., Bjorn M. J., Maxwell E. S. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3211–3215. doi: 10.1073/pnas.75.7.3211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knight D. A., Finck-Barbançon V., Kulich S. M., Barbieri J. T. Functional domains of Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1995 Aug;63(8):3182–3186. doi: 10.1128/iai.63.8.3182-3186.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kudoh I., Wiener-Kronish J. P., Hashimoto S., Pittet J. F., Frank D. Exoproduct secretions of Pseudomonas aeruginosa strains influence severity of alveolar epithelial injury. Am J Physiol. 1994 Nov;267(5 Pt 1):L551–L556. doi: 10.1152/ajplung.1994.267.5.L551. [DOI] [PubMed] [Google Scholar]
  18. Kulich S. M., Frank D. W., Barbieri J. T. Expression of recombinant exoenzyme S of Pseudomonas aeruginosa. Infect Immun. 1995 Jan;63(1):1–8. doi: 10.1128/iai.63.1.1-8.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kulich S. M., Frank D. W., Barbieri J. T. Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun. 1993 Jan;61(1):307–313. doi: 10.1128/iai.61.1.307-313.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kulich S. M., Yahr T. L., Mende-Mueller L. M., Barbieri J. T., Frank D. W. Cloning the structural gene for the 49-kDa form of exoenzyme S (exoS) from Pseudomonas aeruginosa strain 388. J Biol Chem. 1994 Apr 8;269(14):10431–10437. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Liu P. V. 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. 1966 Oct;116(4):481–489. doi: 10.1093/infdis/116.4.481. [DOI] [PubMed] [Google Scholar]
  23. Nicas T. I., Frank D. W., Stenzel P., Lile J. D., Iglewski B. H. Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. Eur J Clin Microbiol. 1985 Apr;4(2):175–179. doi: 10.1007/BF02013593. [DOI] [PubMed] [Google Scholar]
  24. Nicas T. I., Iglewski B. H. Contribution of exoenzyme S to the virulence of Pseudomonas aeruginosa. Antibiot Chemother (1971) 1985;36:40–48. doi: 10.1159/000410470. [DOI] [PubMed] [Google Scholar]
  25. Nicas T. I., Iglewski B. H. Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun. 1984 Aug;45(2):470–474. doi: 10.1128/iai.45.2.470-474.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ohman D. E., Sadoff J. C., Iglewski B. H. Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun. 1980 Jun;28(3):899–908. doi: 10.1128/iai.28.3.899-908.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Olson J. C., Hamood A. N., Vincent T. S., Beachey E. H., Iglewski B. H. Identification of functional epitopes of Pseudomonas aeruginosa exotoxin A using synthetic peptides and subclone products. Mol Immunol. 1990 Oct;27(10):981–993. doi: 10.1016/0161-5890(90)90121-f. [DOI] [PubMed] [Google Scholar]
  28. Olson J. C., Ohman D. E. Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions. J Bacteriol. 1992 Jun;174(12):4140–4147. doi: 10.1128/jb.174.12.4140-4147.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rosqvist R., Magnusson K. E., Wolf-Watz H. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 1994 Feb 15;13(4):964–972. doi: 10.1002/j.1460-2075.1994.tb06341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Straley S. C., Skrzypek E., Plano G. V., Bliska J. B. Yops of Yersinia spp. pathogenic for humans. Infect Immun. 1993 Aug;61(8):3105–3110. doi: 10.1128/iai.61.8.3105-3110.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Woods D. E., Que J. U. Purification of Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1987 Mar;55(3):579–586. doi: 10.1128/iai.55.3.579-586.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Woods D. E., Sokol P. A. Use of transposon mutants to assess the role of exoenzyme S in chronic pulmonary disease due to Pseudomonas aeruginosa. Eur J Clin Microbiol. 1985 Apr;4(2):163–169. doi: 10.1007/BF02013591. [DOI] [PubMed] [Google Scholar]
  34. Xiao B., Smerdon S. J., Jones D. H., Dodson G. G., Soneji Y., Aitken A., Gamblin S. J. Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways. Nature. 1995 Jul 13;376(6536):188–191. doi: 10.1038/376188a0. [DOI] [PubMed] [Google Scholar]
  35. Yahr T. L., Barbieri J. T., Frank D. W. Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J Bacteriol. 1996 Mar;178(5):1412–1419. doi: 10.1128/jb.178.5.1412-1419.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yahr T. L., Hovey A. K., Kulich S. M., Frank D. W. Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene. J Bacteriol. 1995 Mar;177(5):1169–1178. doi: 10.1128/jb.177.5.1169-1178.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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