Graduation Year

2008

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

Andrew Cannons, Ph.D.

Co-Major Professor

Huntington Potter, Ph.D.

Keywords

PCR, Ribotyping, MLST, Proteomics, Growth curves

Abstract

Vibrio parahaemolyticus is a leading cause of seafood-borne illness with a newly emerged pandemic strain. Previous studies compared the pandemic and non-pandemic strains to understand the evolution of the pandemic strain but no definitive explanation for its emergence has been discovered. This study investigated the molecular characteristics of the pandemic strain and growth characteristics at different temperatures. The hypothesis tested was that pandemic strains of V. parahaemolyticus have modifications to their proteome that give a selective advantage over the other V. parahaemolyticus strains at temperatures normally encountered in the environment. Molecular typing techniques; automated ribotyping, pandemic specific PCR and multilocus sequence typing (MLST), were compared to determine the best method for pandemic strain determination. MLST was the best method because it was the most informative and accurate.

Furthermore, nine Florida outbreak strains were identified as pandemic. Using representatives of both strains, growth curves were produced at four temperatures. The five pandemic strains had a significantly faster growth rate at 12°C than five non-pandemic strains. Temperature specific proteomic comparisons were completed using liquid chromatography followed by tandem mass spectroscopy. The proteome differences between these two groups at 12°C included three proteins (DnaA, DnaJ-related protein and DnaK-related protein) with functions related to cold stress. DnaA was expressed in the non-pandemic strain and not the pandemic strain, while the reverse was true for DnaJ-related and DnaK-related proteins. Western blot analysis and LC-MS/MS analysis on additional strains did not support the initial LC-MS/MS results. Growth studies using expression recombinants were employed to investigate these proteins on growth at 12°C.

The overexpression of DnaA and DnaJ-related proteins did not significantly alter the growth rates compared to the control strain, but the overexpression recombinant strains DnaK-5 has a significantly slower growth rate than the control strain, the opposite direction as expected. The pandemic strain grows faster at lower temperatures, but the reason has not been determined. A theory is offered in which the pandemic growth advantage related to regulation of cold stress, leading to a shorter lag phase and faster growth rate after acclimation to the lower temperatures. Further experiments to investigate this theory are discussed.

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