Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medicine

Major Professor

Michael White

Abstract

Apicomplexans are responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii (T. gondii) and the deadliest form of malaria caused by Plasmodium falciparum (P. falciparum). The genomes of these pathogens are now sequenced ushering in a new era of drug development. A major hurdle to exploiting this genome resource is that a large number of the encoded genes are "hypotheticals" and have yet to be characterized. Hypothetical proteins comprise roughly half of the predicted gene complement of T. gondii and P. falciparum and represent the largest class of uniquely functioning proteins in these parasites.

Following the idea that functional relationships can be informed by the timing of gene expression, we devised a strategy to identify the core set of apicomplexan cell division cycling genes with important roles in parasite division, which includes many uncharacterized proteins. We assembled an expanded list of orthologs from the T. gondii and P. falciparum genome sequences (2781 putative orthologs), compared their mRNA profiles during synchronous replication, and sorted the resulting set of dual cell cycle regulated orthologs (744 total) into protein pairs conserved across many eukaryotic families versus those unique to the Apicomplexa. The analysis identified more than 100 ortholog gene pairs with unknown function in T. gondii and P. falciparum that displayed co-conserved mRNA abundance, dynamics of cyclical expression and similar peak timing that spanned the complete division cycle in each parasite. The unknown cyclical mRNAs encoded a diverse set of proteins with a wide range of mass and showed a remarkable conservation in the internal organization of ordered versus disordered structural domains. A representative sample of cyclical unknown genes (16 total) was epitope tagged in T. gondii tachyzoites yielding the discovery of new protein constituents of the parasite inner membrane complex, key mitotic structures and invasion organelles. These results demonstrate the utility of using gene expression timing and dynamic profile to identify proteins with unique roles in Apicomplexa biology.

Additionally, we selected one of these newly identified membrane proteins to further characterize in both T. gondii and P. falciparum. We named the protein inner membrane complex protein 16 (IMC16) due to its IMC localization however; this protein uniquely preferentially targets the developing daughter IMC early in budding and is completely absent from the mother IMC in dividing parasites. IMC16's membrane association cannot be attributed to an alveolin domain and its partial solubility suggests this protein may need more than post-translational modifications to anchor it into the membrane. Proteomic work to determine possible protein interactions highlight a possible phosphorylation by cyclin dependent protein kinase 1 (CDPK1) in the cytoplasm and dephosphorylation by IMC2a to allow it to associate with the IMC similar to the phosphorylation/dephosphorylation mechanisms used by glideosome associated protein 45 (GAP45) to help associate and anchor the glideosome to the IMC.

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