Date Awarded

2003

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Virginia Institute of Marine Science

Advisor

Peter A. Van Veld

Abstract

In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay (USA) striped bass, Morone saxatilis, are currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. This dissertation characterizes an Nramp gene in this species (MsNramp ) and provide evidence for induction following Mycobacterium exposure. The striped bass MsNramp gene and 554 amino acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein specific 'binding-protein-dependent transport system inner membrane component signature,' three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase-C phosphorylation in the amino- and carboxy termini and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely groups MsNramp with other teleost Nramps, and exhibits high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by RT-PCR. Within one day of injection with Mycobacterium marinum, MsNramp expression in vivo was highly induced (17-fold) in peritoneal exudate cells (PE) relative to controls. Levels of MsNramp were increased three- and six-fold on days three and 15, respectively. Injection with Mycobacterium shottsii resulted in two-, five-, and three-fold increases in gene expression in PE over the time course. In vitro, PE expressed significantly higher levels of MsNramp at 4 and 24 hours post-treatment with live and heat-killed M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 hr and 20 mug/ml LPS. Treatment of PE with LPS caused an increase in intracellular superoxide anion (O2-) levels, whereas treatment with live M. marinum caused a significant depression. Cultured anterior kidney cells responded to LPS with increased O2 - and MsNramp production, but were uninduced or suppressed relative to controls by mycobacteria. This study represents the first report of induction of an Nramp gene by mycobacteria in vivo or in vitro in a poikilothermic vertebrate, and supports reports of teleost Nramp induction by LPS.

DOI

https://dx.doi.org/doi:10.25773/v5-gfkc-6079

Rights

© The Author

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