Proteomics analysis Toxoplasma gene knockout phenotypes

描述（由申请人提供）：顶复合体寄生虫是专性细胞内原生动物，可引起严重的人类疾病，包括疟疾、隐孢子虫病和弓形虫病。这些寄生虫必须侵入宿主细胞才能生存、增殖并引起疾病。越来越多的证据表明，寄生虫分泌蛋白在入侵中起着关键作用。母体R01提案的长期目标是阐明寄生虫分泌细胞器的功能，将弓形虫作为其他顶复合体的模型寄生虫。这些研究将致力于确定抗寄生虫治疗的新靶点。虽然亲本提案只关注MIC2蛋白，但在本R21中，我们计划扩大我们的分析范围，包括两个额外的微素蛋白，MIC5和MIC2相关蛋白（M2AP），它们与MIC2形成异源二聚体。这种扩展将使用几种强大的尖端技术来完成，包括基因敲除，高分辨率分离和质谱鉴定蛋白质，以及蛋白质复合物的亲和纯化。本R21提案的具体目标1是确定MIC5、M2AP或MIC2缺失对弓形虫分泌的所有其他蛋白的表达、分泌、翻译后修饰和靶向性的影响。特异性目标2是确定MIC5的蛋白伴侣，这些蛋白伴侣在MIC5入侵期间负责将MIC5靶向到微基因组，然后到达寄生虫表面。我们预计这个“功能蛋白质组学”项目将揭示以前未知的蛋白质相互作用，这将是定义分泌蛋白在寄生虫入侵中的功能的关键。

DESCRIPTION (provided by applicant): Apicomplexan parasites are obligate intracellular protozoa that cause severe human diseases including malaria, cryptosporidiosis, and toxoplasmosis. These parasites must invade host cells to survive, proliferate, and cause disease. There is mounting evidence that parasite secretory proteins play key roles in invasion. The long-term goal of the parent R01 proposal is to elucidate the function of parasite secretory organelles called micronemes, using Toxoplasma gondii as a model parasite for other Apicomplexa. These studies will be conducted in an effort to identify novel targets for anti-parasitic treatments. While the parent proposal focused exclusively on one protein, MIC2, in this R21 we plan to expand the scope of our analysis to include two additional microneme proteins, MIC5 and MIC2-associated protein (M2AP), which forms a heterodimer with MIC2. This expansion will be accomplished using several powerful, cutting-edge technologies including gene knockouts, high-resolution separation and mass spectrometry identification of proteins, and affinity purification of protein complexes. Specific Aim 1 of this R21 proposal is to define the impact of MIC5, M2AP, or MIC2 gone deletions on the expression, secretion, post-translational modifications, and targeting of all other proteins secreted by T. gondii. Specific Aim 2 is to identify the protein partner(s) for MIC5 that are responsible for targeting MIC5 to the micronemes and then to the parasite surface during invasion. We anticipate that this "functional proteomics" project will reveal previously unknown protein interactions that will be pivotal in defining the functions of secretory proteins in parasite invasion.