Proteomic mapping of differential secretion in Toxoplasma gondii

 描述（由申请方提供）：顶复门寄生虫弓形虫是免疫功能低下患者危及生命的脑炎的病原体，此外，如果先天感染，还可导致各种出生缺陷。与疾病相关的病理起源于快速的细胞内裂解复制周期。裂解复制循环包括宿主细胞侵入、在细胞内空泡中复制和从宿主细胞排出的循环。已经研究了必需的宿主细胞侵入步骤，以其作为新型和特异性药物靶点的潜力。在这个过程中， 存在三种分泌细胞器（微丝、棒状体和致密颗粒）。其中，微线体蛋白对外出、滑动运动和宿主细胞侵入贡献最大。这些不同的功能，导致推测是否有不同的内容，在不同的时间分泌的微线，或微线蛋白的功能是否受寄生虫环境的差异。支持和反对这两种模型的观察比比皆是，但没有压倒性的令人信服的数据来解决这两个方向的争论。贡献的数据，将告知这一重要的辩论，该建议利用微线体的Ca 2+依赖的分泌机制中使用的两个不同的条件突变体的微线体分泌动力学的差异。一种突变体在TgDOC 2中具有温度敏感性（ts）突变（ts-DOC 2），另一种突变体具有由缺乏跨膜结构域的TgFLP等位基因的条件性过表达（配体控制）产生的Ferlin样蛋白TgFLP（DN-FLP）的显性负等位基因。ts-DOC 2根本不分泌微丝，并且将产生不含通过微丝分泌的蛋白质的背景。DN-FLP具有功能性组成型微线体分泌，但在Ca 2 +-离子载体诱导的分泌中受损。这种突变体将促进微线体蛋白之间的分化分泌在不同的点沿着寄生虫的出口-运动-入侵的旅程。因此，这些数据将有助于讨论是否有差异微线蛋白分泌如上所述。将从在允许和限制条件下生长的突变体ts-DOC 2和DN-FLP中收集排泄和分泌抗原（ESA）。我们将应用培养物中氨基酸的稳定同位素标记（SILAC）。与该项目的目标最相关的是，SILAC是定量的，并且允许在允许与限制条件下检测不同突变体之间复杂混合物中蛋白质丰度的微小差异。总的来说，不同分泌突变体与SILAC能力的组合将导致前所未有的ESA分辨率水平，并且将增强我们对差异微线体分泌的信心或反驳差异微线体分泌的可能性。因此，这些将是有价值的数据集弓形虫社区在了解独特的寄生过程中的宿主细胞入侵，和见解可能是合理的药物设计中使用。

 DESCRIPTION (provided by applicant): The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles. The lyic replication cycle is comprised of rounds of host cell invasion, replication in the intracellular vacuole, and egress from the host cell. The essential host cell invasion step has been studied for its potential as a novel and specific drug target. During this process, sequential secretion of three secretory organelles (micronemes, rhoptries, and dense granules) occurs. Of these, it is the microneme proteins that contribute most to egress, gliding motility, and host cell invasion. These diverse functions have led to speculation as to whether there are micronemes with different contents secreted at different times, or whether microneme protein function is regulated by differences in parasite environment. Observations in support and against both models abound but there is no overwhelmingly convincing data to settle the argument in either direction. To contribute data that will inform this important debate, this proposal exploits the differential microneme secretion kinetics of two different conditional mutants in the Ca2+-dependent secretion machinery used by the micronemes. One mutant has a temperature sensitive (ts) mutation in TgDOC2 (ts-DOC2), and the other harbors a dominant negative allele of Ferlin-like protein TgFLP (DN-FLP) generated by conditional overexpression (ligand-controlled) of a TgFLP allele lacking the transmembrane domain. ts-DOC2 secretes no micronemes at all and will yield a background free of proteins secreted through the micronemes. DN-FLP has functional constitutive microneme secretion but is impaired in Ca2+-ionophore induced secretion. This mutant will facilitate differentiation between microneme proteins secreted at different points along the parasite's egress-motility-invasion journey. Thus, these data will contribute to the discussion of whether or not there is differential microneme protein secretion as outlined above. Excretory and secretory antigens (ESA) will be collected from mutant ts-DOC2 and DN-FLP grown under both the permissive and restrictive conditions. We will apply Stable Isotope Labeling by Amino Acids in Culture (SILAC). Most pertinent to the goals of this project, SILAC is quantitative and permits the detection of minor differences in protein abundance in complex mixtures across different mutants and under permissive versus restrictive conditions. Overall, the combination of different secretion mutants with the power of SILAC will result in an unprecedented level of ESA resolution and will either boost our confidence in, or disprove the possibility of differential microneme secretion. Hence, these will be valuable data sets for the Toxoplasma community in understanding the uniquely parasitic process of host cell invasion, and the insights could be of use in rational drug design.