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

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。与疾病相关的病理学起源于快速的裂解细胞内复制周期，导致广泛的组织损伤。由于寄生虫的复制只发生在哺乳动物宿主的细胞内，宿主细胞入侵和输出的相关过程标志着致病过程中的必要步骤。已经确定，侵袭和外泄的信号通路围绕细胞质内钙离子浓度的升高而进行。通过药理学研究，两种小的钙结合蛋白，钙调蛋白(CaM；4个EF-Hands)和钙调神经磷酸酶(CaM，调节亚基有4个EF-Hands)与激活出口/入侵有关。此外，由三个中心素(Cen1-3)组成的一组相关的4-EF-Hand蛋白是细胞分裂机制的关键组件：中心体复制中的Cen1和Cen3是有丝分裂和协调异常细胞分裂过程所必需的；Cen2收缩子寄生虫分离所需的基本复合体。本质上，这个EF-Hand蛋白家族在致病的定义步骤中起着关键作用：侵袭/排出和细胞分裂。然而，尽管该蛋白家族在发病机制中起着关键作用，但人们从未对其进行全面的研究。为了填补这一空白，研究人员在基因组中总共确定了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发挥了额外的功能。初步亚细胞 这些蛋白质在整个裂解周期中的定位动态支持这一假说，并允许假定分配在细胞分裂或入侵/出口中的角色。建议完成对整个家庭的这些初步研究，然后对十个优先考虑的候选人进行有条件的基因敲除。为了说明这种方法的威力，这些实验将 首次直接解决CaM或钙调神经磷酸酶在出口/入侵中的作用，目前只有间接的药理学数据可用。此外，利用Cen2和CaM作为先导，将直接剖析钙结合在侵袭或细胞分裂中起关键作用的家庭成员的作用。预计这些研究将揭开Cen2在弓形虫细胞分裂和/或细胞骨架结构中的神秘作用。总之，这项对一个研究较少的基因家族的研究有望为顶丛寄生虫发病机制的两个关键方面提供关键的见解。

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.