Analyzing the dynamics of metabolic responses in Perkinsus marinus in pathways related to oxidative stress

Abstract

Research and Data

Teaching and Resources

• Perkinsus marinus is a pathogenic protist that poses a serious threat to oyster populations.We are interested in analyzing metabolic pathways in P. marinus that balance energy demands in the face of internal and external oxidative stress pressures. Specifically we interested in the enzyme cytoplasmic aconitase, which in addition to participation in these events, may also be involved in regulating citrate utilization. Curiously, an extensive search of the P. marinus genomic database has shown that only the cytosolic, and not the mitochondrial, orthologue is present in this organism. This raises the fascinating question that either this protein has been targeted to the mitochondria, or that there is simply no functioning aconitase in the mitochondria. The RNA seq experiment associated with this project is aimed at analyzing the dynamics of metabolic responses in P. marinus, particularly in pathways related to oxidative stress. We hypothesize a key role for c-aconitase in regulating this pathway, as its isocitrate product is the substrate of a subsequent dehydrogenase activity that increases NADPH production. NADPH is subsequently used in the synthesis of glutathione, a key antioxidant regulator. Differential analysis of gene expression under normal, and oxidatively stressed, growth conditions will enable a snapshot of metabolic responses in this protist parasite. As aconitase is a core TCA cycle enzyme, this project will also yield insights into conserved responses across all kingdoms of life.

  Our straightforward question is to ascertain what metabolic genes are turned on and off during oxidative stress. In addition, it is known that P. marinus is resistant to killing by H2O2, so this study may yield clues as to how this parasite is able to resist oxidative stress. Finally, cytosolic aconitase is regulated by protein kinase C (PKC) in mammalian systems, altering its enzymatic function. A conserved PKC phosphorylation site is present in the P. marinus aconitase, and several PKC orthologues are present in the P. marinus genome. Thus, beyond the various enzymatic pathways that are almost certain to be differentially regulated, we are also very curious to determine what signaling protein orthologues will be stimulated during oxidative stress.

• RNA will be generated from cells grown under two different growth regimens. Growth condition #1 will use a standard growth media that is used to cultivate cells, and in which we were able to generate RNA for a study on spliced leader RNA from P. marinus. Growth media #2 will be contain H2O2 at a concentration of 10 mM, which is known to induce oxidative stress in cells. Our straightforward question is to ascertain what metabolic genes are turned on and off during these distinct conditions. In addition, it is known that P. marinus is resistant to killing by H2O2, so this study may yield clues as to how this parasite is able to resist oxidative stress. Finally, cytosolic aconitase is regulated by protein kinase C (PKC) in mammalian systems, altering its enzymatic function. A conserved PKC phosphorylation site is present in the P. marinus aconitase, and several PKC orthologues are present in the P. marinus genome. Thus, beyond the various enzymatic pathways that are almost certain to be differentially regulated, we are also very curious to determine what signaling protein orthologues will be stimulated during oxidative stress.

• Courses

  At UMES I am responsible for teaching students at all levels, including introductory biology for majors, a sophomore level course in genetics, and a junior/senior level course in cell biology. In spring of 2016 I will be teaching a new course in molecular genetics. I will assign a semester project based upon this proposed study. The laboratory portion of the course will include both training in bioinformatics methods and in various cloning procedures. The RNAseq analysis will be integrated into the course through a unit describing bioinformatics approaches.