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Volume 146, Issue 1

Use of green fluorescent protein (GFP) to study the invasion pathways of in and fish models Free

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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Keyword(s): carp epithelial cells , Edwardsiella tarda , EPC, epithelioma papillosum of carp , GFP , GFP, green fluorescent protein , infection kinetics and MAPK, mitogen-activated protein kinase
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2000-01-01
2024-04-23
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Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models
Microbiology 146, 7 (2000); https://doi.org/10.1099/00221287-146-1-7
/content/journal/micro/10.1099/00221287-146-1-7
/content/journal/micro/10.1099/00221287-146-1-7
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