Apicomplexan parasites are important human pathogens, and cause diseases ranging from life-long asymptomatic infections with Toxoplasma gondii in about a quarter of the world's population to nearly a million deaths annually due to malaria. To decipher their biology and treat the diseases they cause, we must understand the signaling pathways unique to these successful pathogens. Calcium-dependent protein kinases (CDPKs) are attractive targets for intervention because they are conserved among apicomplexans, absent from the genomes of their animal hosts, and essential for the parasite life cycle. Prior work has shown that CDPKs regulate various processes necessary during the T. gondii life cycle, including the calcium-regulated secretion of specialized organelles required for motility. Although we have identified key enzymes responsible for phosphorylation in T. gondii, we know little about the substrates, and even less about the consequences of these modifications for parasite entry, survival and release from the infected host cell. The proposed study will map essential signaling pathways regulated by apicomplexan CDPKs and inform their potential as therapeutic targets. The three specific aims of this proposal will address different aspects of CDPK biology, by identifying the role of individual kinases, characterizing the substrates they regulate, and determining the function of these substrates.
The first aim uses a chemical-genetic strategy established by the applicant to specifically inhibit and study the function of two CDPKs in the parasite life cycle, and extends this strategy to the four remaining members of the kinase family. These experiments will allow us to compare the cellular processes regulated by each of the conserved CDPKs in T. gondii.
The second aim exploits our ability to label and identify the targets of specific parasite kinases, to map the substrates of two CDPKs previously shown to be essential for parasite entry and exit from host cells.
The final aim will use quantitative mass spectrometry and genetic manipulation-guided by CDPK targets we already identified and those identified in the second aim-to measure phosphorylation changes in vivo and determine the function of selected CDPK targets. Together the second and third aims will characterize components of the pathways regulated by CDPKs, and establish the molecular basis for their essential function. The goal of this study is to map essential signaling networks regulated by apicomplexan CDPKs and inform their potential as therapeutic targets. Newly identified substrates of individual kinases are likely novel components of these pathways. This is relevant because we don't know the function of ~40% of apicomplexan proteins or the pathways in which they participate. Furthermore, this study provides the basis for comparing CDPK functions across apicomplexans, to uncover how this kinase family regulates the behavior of different organisms.
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.
|Rothenberg, Daniel A; Gordon, Elizabeth A; White, Forest M et al. (2016) Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles. Methods Mol Biol 1355:71-84|
|Sidik, Saima M; Hortua Triana, Miryam A; Paul, Aditya S et al. (2016) Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling. J Biol Chem 291:9566-80|
|Sidik, Saima M; Huet, Diego; Ganesan, Suresh M et al. (2016) A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell 166:1423-1435.e12|
|Swee, Lee Kim; Lourido, Sebastian; Bell, George W et al. (2015) One-step enzymatic modification of the cell surface redirects cellular cytotoxicity and parasite tropism. ACS Chem Biol 10:460-5|
|Lourido, Sebastian; Moreno, Silvia N J (2015) The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium 57:186-93|
|Gold, Daniel A; Kaplan, Aaron D; Lis, Agnieszka et al. (2015) The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole. Cell Host Microbe 17:642-52|
|Ingram, Jessica R; Knockenhauer, Kevin E; Markus, Benedikt M et al. (2015) Allosteric activation of apicomplexan calcium-dependent protein kinases. Proc Natl Acad Sci U S A 112:E4975-84|
|Sidik, Saima M; Hackett, Caroline G; Tran, Fanny et al. (2014) Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9:e100450|