HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ling, S. H. M. Articles by Leung, K. Y. Search for Related Content PubMed PubMed Citation Articles by Ling, S. H. M. Articles by Leung, K. Y. Agricola Articles by Ling, S. H. M. Articles by Leung, K. Y.

Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models

Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260¹

Author for correspondence: K. Y. Leung. Tel: +65 8747835. Fax: +65 7792486. e-mail: dbslky{at}nus.edu.sg

Edwardsiella tarda is a fish pathogen that causes systemic infections in many food and ornamental fish. E. tarda 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 E. tarda in vitro and in vivo. Epithelioma papillosum of carp (EPC) was used as the first model. Virulent and avirulent E. tarda strains were found to adhere to and invade EPC cells. Interactions between E. tarda 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 E. tarda 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 E. tarda and its hosts.

Keywords: Edwardsiella tarda, GFP, carp epithelial cells, infection kinetics

Abbreviations: EPC, epithelioma papillosum of carp; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase

This article has been cited by other articles:

				J. Zheng, N. Li, Y. P. Tan, J. Sivaraman, Y.-K. Mok, Z. L. Mo, and K. Y. Leung EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD Microbiology, June 1, 2007; 153(6): 1953 - 1962. [Abstract] [Full Text] [PDF]

				M. Du, J. Chen, X. Zhang, A. Li, Y. Li, and Y. Wang Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate Appl. Envir. Microbiol., February 15, 2007; 73(4): 1349 - 1354. [Abstract] [Full Text] [PDF]

				P. S. Srinivasa Rao, T. M. Lim, and K. Y. Leung Functional Genomics Approach to the Identification of Virulence Genes Involved in Edwardsiella tarda Pathogenesis Infect. Immun., March 1, 2003; 71(3): 1343 - 1351. [Abstract] [Full Text] [PDF]

				Y. P. Tan, Q. Lin, X. H. Wang, S. Joshi, C. L. Hew, and K. Y. Leung Comparative Proteomic Analysis of Extracellular Proteins of Edwardsiella tarda Infect. Immun., November 1, 2002; 70(11): 6475 - 6480. [Abstract] [Full Text] [PDF]

				P. S. S. Rao, T. M. Lim, and K. Y. Leung Opsonized Virulent Edwardsiella tarda Strains Are Able To Adhere to and Survive and Replicate within Fish Phagocytes but Fail To Stimulate Reactive Oxygen Intermediates Infect. Immun., September 1, 2001; 69(9): 5689 - 5697. [Abstract] [Full Text] [PDF]

				M. Zhao, M. Yang, E. Baranov, X. Wang, S. Penman, A. R. Moossa, and R. M. Hoffman Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals PNAS, July 24, 2001; (2001) 161275798. [Abstract] [Full Text] [PDF]

				J. A. Mathew, Y. P. Tan, P. S. Srinivasa Rao, T. M. Lim, and K. Y. Leung Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity Microbiology, February 1, 2001; 147(2): 449 - 457. [Abstract] [Full Text]