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Dissecting Essential Signaling Pathways in Apicomplexan Parasites


Apicomplexan parasites are important human pathogens, and cause diseases ranging from life-longasymptomatic infections with Toxoplasma gondii in about a quarter of the world's population to nearly a milliondeaths annually due to malaria. To decipher their biology and treat the diseases they cause, we mustunderstand the signaling pathways unique to these successful pathogens. Calcium-dependent protein kinases(CDPKs) are attractive targets for intervention because they are conserved among apicomplexans, absentfrom the genomes of their animal hosts, and essential for the parasite life cycle. Prior work has shown thatCDPKs regulate various processes necessary during the T. gondii life cycle, including the calcium-regulatedsecretion of specialized organelles required for motility. Although we have identified key enzymes responsiblefor phosphorylation in T. gondii, we know little about the substrates, and even less about the consequences ofthese modifications for parasite entry, survival and release from the infected host cell. <p/>The proposed study will map essential signaling pathways regulated by apicomplexan CDPKs and inform theirpotential as therapeutic targets. The three specific aims of this proposal will address different aspects of CDPKbiology, by identifying the role of individual kinases, characterizing the substrates they regulate, anddetermining the function of these substrates. <p/>The first aim uses a chemical-genetic strategy established by theapplicant to specifically inhibit and study the function of two CDPKs in the parasite life cycle, and extends thisstrategy to the four remaining members of the kinase family. These experiments will allow us to compare thecellular processes regulated by each of the conserved CDPKs in T. gondii. The second aim exploits our abilityto label and identify the targets of specific parasite kinases, to map the substrates of two CDPKs previouslyshown to be essential for parasite entry and exit from host cells. The final aim will use quantitative massspectrometry and genetic manipulation-guided by CDPK targets we already identified and those identified inthe second aim-to measure phosphorylation changes in vivo and determine the function of selected CDPKtargets. Together the second and third aims will characterize components of the pathways regulated by CDPKs,and establish the molecular basis for their essential function. <p/>The goal of this study is to map essential signaling networks regulated by apicomplexan CDPKs and informtheir potential as therapeutic targets. Newly identified substrates of individual kinases are likely novelcomponents of these pathways. This is relevant because we don't know the function of ~40% of apicomplexanproteins or the pathways in which they participate. Furthermore, this study provides the basis for comparingCDPK functions across apicomplexans, to uncover how this kinase family regulates the behavior of differentorganisms.

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PUBLIC HEALTH RELEVANCE: The proposed study will investigate essential signaling pathways in Toxoplasma gondii, which is an important opportunistic pathogen and NIAID category B Biodefense agent. T. gondii also serves as a model for parasites that are more difficult to study, such as Plasmodium (malaria) and Cryptosporidium, which share the pathways that are the focus of this work. These pathways are attractive pharmacological targets, because they are absent from humans, and our work will serve to characterize their function and determine their therapeutic potential.

Lourido, Sebastian
Whitehead Institute for Biomedical Research
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