| The toxic dinoflagellate, Karenia brevis, forms nearly annual blooms in the Gulf of Mexico that persist for many months in coastal waters, causing extensive marine animal mortalities and human health impacts. The molecular mechanisms that contribute to cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. The studies presented in this dissertation investigate the existence and involvement of a programmed cell death-like (PCD-like) pathway in the demise of K. brevis cultures following oxidative stress and chronological aging. Firstly, to gain an understanding of the molecular processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out and identified extensive transcriptomic remodeling during the transition into stationary phase indicative of a shift in the metabolic and signaling requirements for survival in a quiescent non-dividing phase. To better understand the connection between the transcriptomic context identified in the microarray study and the presence of a PCD-like pathway in K. brevis, hallmark morphological and biochemical changes (DNA fragmentation, caspase-like activity, and caspase 3-like protein expression) were used to define PCD-like morphological changes following chronological aging and oxidative stress. Targeted in silico bioinformatic mining was used to identify enzymes potentially responsible for the activities observed, as well as the substrates. Finally, K. brevis S-adenosylmethionine synthetase (KbAdoMetS), a putative caspase substrate predicted from the bioinformatics screen, was examined using MALDI-TOF MS to confirm the validity of the bioinformatics approach. Taken together, this work identified that K. brevis contains morphological changes indicative of a caspase-dependent PCD-like pathway and that KbAdoMetS is a caspase 3-like substrate. Finally, we sought to characterize the presence of metacaspases in Karenia brevis, and specifically evaluated the role of metacaspase 1 (KbMC1) during chronological aging and death in culture. Immunocytochemistry, subcellular fractionation, and western blotting results using a custom KbMC1 peptide antibody indicate that KbMC1 may be involved in PCD-like execution through its chloroplastic localization with proposed interactions with the photosynthetic machinery. This study provides the first comprehensive investigation of the molecular processes regulating chronological aging and execution of PCD-like death in a toxic dinoflagellate. |
