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Oysters & the
Chesapeake

 
Oyster Research and Restoration in U.S. Coastal Waters: Strategies for the Future
September 8-9, 2003 - Annapolis, Maryland

Abstracts
Workgroup: Frontiers in Disease Research

Taxonomic and Genetic Characterization of Perkinsus marinus. Development of Mutagenesis and Gene transfer Systems with Application to Therapeutic Strategies.

Principal Investigator(s):
Gerardo R. Vasta, Center of Marine Biotechnology, Baltimore, MD, vasta@umbi.umd.edu

Funding Period: 1997-99

This project saw significant advances in the development of both heterologous (yeast and bacteria) expression of P. marinus SOD genes, as well as progress towards the goal of directly transforming P. marinus with expression constructs.
Development of tools in this initiative benefited from advances made in previous and concurrently SeaGrant -funded projects (R-ODR/7).
  • Improved reporter genes (luciferase, G418 resistance, and enhanced GFP) were tested, and the latter (GFP) used to make fusions with an endogenous P. marinus promoter (PmSOD1, which is expressed in all cells).
  • We obtained direct evidence that we have introduced DNA into P. marinus trophozoites by electroporation. This was the result of modification of culture conditions prior to electroporation as well as optimization of electroporation conditions.
  • The information obtained in this initiative led to the construction of a high quality genomic library, from which we have obtained full genomic sequences of more than a half dozen genes.
  • Solid media growth conditions were established that should allow for the selection of P. marinus mutants in metabolic and oxidative stress pathways.

IMPACTS and/or BENEFITS:
Progress toward the construction of transgenic P. marinus will be of great value in many respects, advancing the objective of developing anti-parasite therapies or management strategies. In addition to the tremendous boost to basic research into cell proliferation, structure, and parasite life cycle, it will accelerate discoveries about infection/invasion mechanisms and virulence factors. It will allow the direct testing of hypotheses about virulence factors and infection mechanisms.
The crucial experiments P. marinus transformation will make possible include:
  • Creation of mutants in pathways of interest to test their effect on disease.
  • Promoter-trapping of genes expressed during infection processes.
The value of the genomic library made possible by this project is being repaid many times over as each new gene is clones and characterized.

PROJECT PUBLICATIONS:

Coss, C.A., Robledo, J.A.F., Vasta, G.R. 2001. Fine structure of clonally propagated in vitro life stages of a Perkinsus sp. isolated from Baltic clam Macoma balthica. Journal of Eukaryotic Microbiology. 48: 38-51.

Robledo, J.A.F., Coss, C.A., Vasta, G.R. 2000. Characterization of the NTS, SSU and ITS from the RNA locus of Perkinsus atlanticus from Clams (Ruditapes decussatus) and development of a species-specific PCR-based diagnostic assay. Jourbal of Parasitology. 86: 972-978.

Robledo, J.A.F., Coss, C.A., Marsh, A.G., Wright, A.C., Vasta, G.R. 1999. Genetic nucleotide sequence variability in the non-transcribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus. Journal of Parasitology. 85: 650-656.

UM-SG-TS-2003-01 www.mdsg.umd.edu
   
This publication was supported by funds from
the NOAA National Sea Grant College Program and the
Maryland and Virginia Sea Grant College Programs
[Maryland Sea Grant]
 [NOAA]
 [Virginia Sea Grant]

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