1887

Abstract

is a fish pathogen that causes systemic infections in many food and ornamental fish. PPD130/91 and PPD125/87 were selected as representatives of the virulent and avirulent groups, respectively, from eight fish isolates, and transformed with plasmids encoding either green fluorescent protein (pGFPuv) or blue fluorescent protein (pBFP2). Two host models were used to study the invasion pathway of and . Epithelioma papillosum of carp (EPC) was used as the first model. Virulent and avirulent strains were found to adhere to and invade EPC cells. Interactions between and host cells examined under confocal microscopy and intracellular growth were followed at different time points. Bacterial internalization of PPD130/91 and PPD125/87 involved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization. Confocal studies revealed co-localization of polymerized actin with bacteria. Staurosporine, a protein kinase C inhibitor, accelerated internalization of PPD125/87, whereas PD098059, a mitogen-activated protein kinase (MAPK) kinase inhibitor prevented internalization of PPD130/91. In the second model, blue gourami were infected with intramuscularly. Mortalities were observed in PPD130/91(pGFPuv)-infected fish with high bacterial numbers detectable in all organs. PPD125/87(pBFP2)-infected fish did not die and the bacterial population decreased over time. Mixed infections comprised of both PPD130/91(pGFPuv) and PPD125/87(pBFP2), where inoculum size was similar to the single infections, caused mortalities in fish. High bacterial populations were noted only in the fish body muscle. The PPD125/87(pBFP2) population in the fish decreased after 5 d. The number of PPD130/91(pGFPuv) also decreased in the fish organs, except for continued high growth in the body muscle. Histology revealed necrosis of the tissue (body muscle and liver) and fluorescent bacteria in fish that were infected with PPD130/91(pGFPuv) but not with PPD125/87(pBFP2). This study showed that fluorescent proteins are a useful tool for investigating bacterial host cell infection, and information elucidated here sheds new light on the interactions between and its hosts.

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2000-01-01
2020-09-30
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References

  1. Ainsworth A. J., Dexiang C.. 1990; Differences in the phagocytosis of four bacteria by channel catfish neutrophils. Dev Comp Immunol14:201–209[CrossRef]
    [Google Scholar]
  2. Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y.. 1987; Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem262:5592–5595
    [Google Scholar]
  3. Amandi A., Hiu S. F., Rohovec J. S., Fryer J. L.. 1982; Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha). Appl Environ Microbiol43:1380–1384
    [Google Scholar]
  4. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C.. 1994; Green fluorescent protein as a marker for gene expression. Science263:802–805[CrossRef]
    [Google Scholar]
  5. Clerc P., Sansonetti P. J.. 1987; Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect Immun55:2681–2688
    [Google Scholar]
  6. Crameri A., Whitehorn E. A., Tate E., Stemmer W. P. C.. 1995; Improved green fluorescence protein by molecular evolution using DNA shuffling. Nature Biotechnol14:315–319
    [Google Scholar]
  7. Elsinghorst E. A.. 1994; Measurement of invasion by gentamicin resistance. Methods Enzymol236:405–420
    [Google Scholar]
  8. Evelyn T. P. T.. 1996; Infection and disease. In The Fish Immune System – Organism, Pathogen, and Environment pp.339–359Edited by Iwama G., Nakanishi T.. San Diego: Academic Press;
    [Google Scholar]
  9. Farmer J. J., McWhorter A. C.. 1984; Genus X. Edwardsiella. In Bergey’s Manual of Systematic Bacteriology pp.486–491Edited by Holt J. G., Krieg N. R.. Baltimore: Williams & Wilkins;
    [Google Scholar]
  10. Finlay B. B., Cossart P.. 1997; Exploitation of mammalian host cell functions by bacterial pathogens. Science276:718–725[CrossRef]
    [Google Scholar]
  11. Finlay B. B., Falkow S.. 1988; Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie70:1089–1099[CrossRef]
    [Google Scholar]
  12. Finlay B. B., Falkow S.. 1989; Salmonella as an intracellular parasite. Mol Microbiol3:1833–1841[CrossRef]
    [Google Scholar]
  13. Finlay B. B., Falkow S.. 1997; Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev61:136–169
    [Google Scholar]
  14. Finlay B. B., Ruschkowski S., Dedhar S.. 1991; Cytoskeletal rearrangements accompanying Salmonella entry into epithelial cells. J Cell Sci99:283–296
    [Google Scholar]
  15. Hirono I., Tange N., Aoki T.. 1997; Iron-regulated haemolysin gene from Edwardsiella tarda. Mol Microbiol24:851–856[CrossRef]
    [Google Scholar]
  16. Janda J. M., Abbott S. L., Oshiro L. S.. 1991a; Penetration and replication of Edwardsiella spp. in HEp-2 cells. Infect Immun59:154–161
    [Google Scholar]
  17. Janda J. M., Abbott S. L., Kroske-Bystrom S., Cheung W. K. W., Powers C., Kokka R. P., Tamura K.. 1991b; Pathogenic properties of Edwardsiella species. J Clin Microbiol29:1997–2001
    [Google Scholar]
  18. Kubota S. S., Kaige N., Miyazaki T., Miyashita T.. 1981; Histopathological studies on edwardsiellosis of tilapia. I. Natural infection. Bull Fac Fish Mie Univ9:155–165
    [Google Scholar]
  19. Kusuda R., Ishihara S.. 1981; The fate of Edwardsiella tarda bacteria after intramuscular injection of eels, Anguilla japonica. Bull Jpn Soc Sci Fish47:475–479[CrossRef]
    [Google Scholar]
  20. Leung K. Y., Finlay B. B.. 1991; Intracellular replication is essential for the virulence of Salmonella typhimurium. Proc Natl Acad Sci USA88:11470–11474[CrossRef]
    [Google Scholar]
  21. Leung K. Y., Yeap I. V., Lam T. J., Sin Y. M.. 1995; Serum resistance as a good indicator for virulence in Aeromonas hydrophila strains isolated from diseased fish in South-East Asia. J Fish Dis18:511–518[CrossRef]
    [Google Scholar]
  22. Meyer F. P., Bullock G. L.. 1973; Edwardsiella tarda, a new pathogen of channel catfish (Ictalurus punctatus). Appl Microbiol25:155–156
    [Google Scholar]
  23. Nakatsugawa T.. 1983; Edwardsiella tarda isolated from cultured young flounder. Fish Pathol18:99–101[CrossRef]
    [Google Scholar]
  24. Plumb J. A.. 1993; Bacterial Diseases of FishEdited by Bromage N. R., Inglis V., Roberts R. J.. Oxford: Blackwell Scientific Publications;
    [Google Scholar]
  25. Prasher D. C., Eckenrode V. K., Ward W. W., Prendgast F. G., Cormier M. J.. 1992; Primary structure of the Aequorea victoria green-fluorescent protein. Gene111:229–233[CrossRef]
    [Google Scholar]
  26. Quinn F. D., Newman G. W., King C. H.. 1997; In search of virulence factors of human bacterial disease. Trends Microbiol5:20–26[CrossRef]
    [Google Scholar]
  27. Rashid M. M., Nakai T., Muroga K., Miyazaki T.. 1997; Pathogenesis of experimental edwardsiellosis in Japanese flounder Paralichthys olivaceus. Fish Sci63:384–387
    [Google Scholar]
  28. Rasouly D., Lazarovici P.. 1994; Staurosporine induces tyrosine phosphorylation of a 145 kDa protein but does not activate gp140trk in PC12 cells. Eur J Pharmacol269:255–264[CrossRef]
    [Google Scholar]
  29. Reed L. J., Muench H.. 1938; A simple method of estimating fifty per cent end points. Am J Hyg27:493–497
    [Google Scholar]
  30. Rosenshine I., Donnenberg M. S., Kaper J. B., Finlay B. B.. 1992; Signal transduction between enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPEC induces tyrosine phosphorylation of host cell proteins to initiate cytoskeletal rearrangement and bacterial uptake. EMBO J11:3551–3560
    [Google Scholar]
  31. Sae-Oui D., Muroga K., Nakai T.. 1984; A case of Edwardsiella tarda in cultured colored carp, Cyprinus carpio. Fish Pathol19:197–199[CrossRef]
    [Google Scholar]
  32. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  33. Strauss E. J., Ghori N., Falkow S.. 1997; An Edwardsiella tarda strain containing a mutation in a gene with homology to shlB and hpmB is defective for entry into epithelial cells in culture. Infect Immun65:3924–3932
    [Google Scholar]
  34. Suprapto H., Hara T., Nakai T., Muroga K.. 1996; Purification of a lethal toxin of Edwardsiella tarda. Fish Pathol31:203–207[CrossRef]
    [Google Scholar]
  35. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F.. 1986; Staurosporine, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem Biophys Res Commun135:397–402[CrossRef]
    [Google Scholar]
  36. Tan E., Low K. W., Wong W. S. F., Leung K. Y.. 1998; Internalization of Aeromonas hydrophila by fish cells can be inhibited with a tyrosine kinase inhibitor. Microbiology144:299–307[CrossRef]
    [Google Scholar]
  37. Ullah M. A., Arai T.. 1983; Pathological activities of the naturally occurring strains of Edwardsiella tarda. Fish Pathol18:65–70[CrossRef]
    [Google Scholar]
  38. Valdivia R. H., Hromockyj A. E., Monack D., Ramakrishnan L., Falkow S.. 1996; Applications for green fluorescent protein (GFP) in the study of host–pathogen interactions. Gene173:47–52[CrossRef]
    [Google Scholar]
  39. Van Damme L. R., Vandepitte J.. 1984; Isolation of Edwardsiella tarda and Plesiomonas shigelloides from mammals and birds in Zaire. Rev Elev Med Vet Pays Trop37:145–151
    [Google Scholar]
  40. Wakabayashi H., Egusa S.. 1973; Edwardsiella tarda (Paracolobactrum anguillmortiferum) associated with pond-cultured eel disease. Bull Jpn Soc Sci Fish39:931–936[CrossRef]
    [Google Scholar]
  41. Wang X. H., Oon H. L., Ho G. W. P., Wong W. S. F., Lim T. M., Leung K. Y.. 1998; Internalization and cytotoxicity are important virulence mechanisms in vibrio–fish epithelial cell interactions. Microbiology144:2987–3002[CrossRef]
    [Google Scholar]
  42. White F. H., Simpson C. F., Williams L. E.. 1973; Isolation of Edwardsiella tarda from aquatic animal species and surface waters in Florida. J Wildl Dis9:204–207[CrossRef]
    [Google Scholar]
  43. Wolf K., Mann J. A.. 1980; Poikilotherm vertebrate cell lines and viruses, a current listing for fishes. In Vitro16:168–179[CrossRef]
    [Google Scholar]
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