Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

Winter 1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Laurence D Sibley

Abstract

Apicomplexans are protozoan parasites of animals, which must penetrate host cells to find a niche in which to replicate. To humans, they represent the most ubiquitous and deadliest eukaryotic pathogens, including Toxoplasma gondii and Plasmodium spp., the etiological agents of toxoplasmosis and malaria, respectively. A common mechanism enables these parasites to penetrate biological barriers and invade host cells actively, through a process termed gliding motility. This type of motility, unique to apicomplexans, relies on the directional translocation of adhesins via an actomyosin motor complex anchored in a vesicular network underlying the plasma membrane of the parasite. A variety of stimuli can trigger calcium increases in the parasite cytoplasm initiating motion, in part by regulating the secretion of adhesins from specialized organelles, called micronemes. Our studies investigate how these calcium signals are transduced to regulate T. gondii motility. Apicomplexans, like other chromalveolates and plants, possess calcium-dependent protein kinases: CDPKs) that are directly activated by calcium binding and have been proposed to participate in the transduction of calcium signals. A number of CDPKs are conserved among apicomplexans, and showed distinct subcellular localizations upon tagged expression in T. gondii, consistent with roles in different calcium-activated cellular pathways. Using a combination of chemical and genetic approaches we demonstrated that two of the conserved CDPKs are required for motility at different stages in the T. gondii life cycle. Generating a conditional knockout, we showed that TgCDPK1 is required for microneme secretion during egress and invasion. The essential role of this kinase was further supported through the use of bulky pyrazolo [3,4-d] pyrimidine: PP) analogues that inhibit TgCDPK1, and mirrored the effects of the conditional knockout. The specificity of these compounds is conferred by the expansion of the ATP-binding pocket in TgCDPK1 caused by the presence of a glycine at a key position called the `gatekeeper', a feature unique among active parasite kinases. Mutating this residue to a methionine made TgCDPK1 resistant to the inhibitors, and enabled us to mutate the gatekeeper residue of a related kinase, TgCDPK3, rendering it sensitive to inhibition by PP analogues. This chemical-genetic strategy allowed us to implicate TgCDPK3 in the initiation of motility during egress, but demonstrate that its function is dispensable during invasion. Together, these observations provide the first evidence that related CDPKs regulate distinct signaling pathways, which distinguish the signaling events governing motility during egress and invasion by T. gondii. We also attempted to further understand the role of TgCDPK1 by examining its cellular targets. This process was also facilitated by the atypical ATP-binding pocket of TgCDPK1, which was able to bind bulky ATP analogues that allowed us to track its direct targets. This approach has allowed us to identify a number of putative TgCDPK1 targets. One such target, a dynamin-related protein, is phosphorylated in vivo in a CDPK1-dependent manner, consistent with a role in the regulation of motility. Together these observations provide a foundation for further characterization of CDPK signaling and the regulation of parasite motility. In particular, the chemical-genetic approaches adapted to parasites in these studies, represent a systematic means to dissecting these essential pathways in apicomplexans.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7QZ280G

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