Organization of Toxoplasma invasion and cell division by EF-hand proteins

描述（由申请人提供）：弓形虫是免疫功能低下患者危及生命的脑炎病原体，此外，如果感染是先天性的，还可能导致多种出生缺陷。与疾病相关的病理起源于快速的细胞内裂解复制周期，引起广泛的组织损伤。由于寄生虫的复制只发生在哺乳动物宿主的一个细胞内，因此宿主细胞的入侵和退出的相关过程标志着其发病机制的重要步骤。它已经坚定地确立了信号通路的入侵和出口围绕细胞质Ca2+浓度的上升。两个小的Ca2+结合蛋白，钙调蛋白（CaM； 4个EF-hand）和钙调磷酸酶（调节亚基有4个EF-hand），通过药理学研究与出口/入侵的激活有关。此外，一组包含3个中心体（Cen1-3）的含有4-EF-hand的小蛋白质是细胞分裂机制的关键组成部分：中心体复制中的Cen1和Cen3是有丝分裂和不寻常的细胞分裂过程协调所必需的；Cen2在子寄生分离所需的基础复合体的收缩中起作用。从本质上讲，EF-hand蛋白家族在确定发病机制的步骤中起着关键作用：侵袭/输出和细胞分裂。然而，尽管该蛋白家族在发病机制中起着关键作用，但尚未对其进行全面研究。为了填补这一空白，研究人员在基因组中发现了16种这样的蛋白质，其中大多数根本没有被研究过，但可能在这些过程中执行额外的功能。初步的亚细胞

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, causin extensive tissue damage. Since parasite replication only occurs within a cell from the mammalian host, the related processes of host cell invasion and egress mark essential steps in the pathogenesis. It has been firmly established that the signaling pathways underlying invasion and egress revolve around the cytoplasmic rise of Ca2+ concentration. Two small, Ca2+-binding proteins, calmodulin (CaM; 4 EF-hands) and the calcineurin phosphatase (the regulatory ¿-subunit has 4 EF hands), have been associated with activation of egress/invasion through pharmacological studies. Furthermore, a related group of small, 4-EF-hand containing proteins consisting of three centrins (Cen1-3) are critical components to the cell division machinery: Cen1 and Cen3 in centrosome duplication, required for mitosis and coordination of the unusual cell division process; Cen2 in contraction of the basal complex required for daughter parasite segregation. In essence, this family of EF-hand proteins plays pivotal roles in the defining steps of pathogenesis: invasion/egress and cell division. However this protein family has never been comprehensively studied, despite their critical roles in pathogenesis. To fill this void, the researchers identified a total of 16 such proteins in the genome, most of which have not been studied at all but likely execute additional functions in these processes. Preliminary sub-cellular localization dynamics of these proteins throughout the lytic cycle support this hypothesis and permits putative assignment to roles in cell division or invasion/egress. It is proposed to complete these preliminary studies for the whole family followed by conditional gene knock-outs of the ten prioritized candidates. To illustrate the power of this approach, these experiments will for the first time directly address whether CaM or calcineurin have a role in egress/invasion, for which only indirect pharmacological data is currently available. Furthermore, using the Cen2 and CaM as a pilot, the direct dissection of the role of Ca2+-binding in the family members with critical roles in invasion or cell division will be pioneered. It is anticipated that these studieswill lift the veil on the enigmatic role of Cen2 in Toxoplasma cell division and/or cytoskeleton structure. In conclusion, this study of a poorly studied gene family is expected to provide key insights into two key aspects in apicomplexan parasite pathogenesis.