Proteomic mapping of differential secretion in Toxoplasma gondii

 描述(由申请人提供)：弓形虫寄生虫是免疫功能低下患者中危及生命的脑炎的病原体，此外，如果感染是先天性感染，还可能导致各种出生缺陷。与疾病相关的病理起源于快速的裂解细胞内复制周期。裂解复制循环由几轮宿主细胞入侵、在细胞内液泡中复制和从宿主细胞出口组成。重要的宿主细胞侵袭步骤因其作为一种新的和特异的药物靶点的潜力而被研究。在这个过程中，顺序分泌的 有三个分泌细胞器(微丝、棒状和致密颗粒)。其中，微线体蛋白对出口、滑动运动和宿主细胞入侵贡献最大。这些不同的功能导致人们猜测，是否存在不同时间分泌的不同含量的微线体，或者微线体蛋白的功能是否受寄生虫环境的不同调节。支持和反对这两种模型的观点比比皆是，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。为了给这场重要的辩论提供信息，这一提议利用了两个不同的条件突变体在微线条使用的钙依赖的分泌机制中的差异微线条分泌动力学。一个突变体在TgDOC2(ts-DOC2)上存在温度敏感(Ts)突变，另一个突变体携带一个显性的类Ferlin蛋白负等位基因(dN-FLP)，该等位基因是由缺乏跨膜结构域的TgFLP等位基因条件过表达(配体控制)而产生的。TS-DOC2根本不分泌微线粒，并将产生一个没有通过微线粒分泌的蛋白质的背景。DN-FLP具有功能性结构性微丝分泌功能，但在钙离子载体诱导的分泌过程中受到抑制。这个突变体将促进微线体蛋白的分化，这些微线体蛋白分泌在寄生虫出口-移动-入侵旅程的不同点上。因此，这些数据将有助于讨论是否存在如上所述的差异微线蛋白分泌。从在允许和限制条件下生长的突变体ts-DOC2和dN-FLP中收集排泄和分泌抗原(ESA)。我们将应用培养中的氨基酸稳定同位素标记(SILAC)。与该项目的目标最相关的是，SILAC是定量的，允许在允许和限制的条件下检测不同突变的复杂混合物中蛋白质丰度的微小差异。总体而言，将不同的分泌突变体与SILAC的能力相结合，将导致前所未有的ESA分辨率，并将增强我们对差异微丝分泌的信心，或反驳其可能性。因此，这些将是有价值的数据集，有助于弓形虫群落理解宿主细胞入侵的独特寄生过程，这些见解可能有助于合理的药物设计。

 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.