Identification of TgCBAP, a Novel Cytoskeletal Protein that Localizes to Three Distinct Subcompartments of the Toxoplasma gondii Pellicle

The cytoskeletons of Toxoplasma gondii and related apicomplexan parasites are highly polarized, with apical and basal regions comprised of distinct protein complexes. Components of these complexes are known to play important roles in parasite shape, cell division, and host cell invasion. During an effort to discover the biologically relevant target of a small-molecule inhibitor of T. gondii invasion (Conoidin A), we discovered a novel cytoskeletal protein that we named TgCBAP (Conserved Basal Apical Peripheral protein). Orthologs of TgCBAP are only found in the genomes of other apicomplexans; they contain no identifiable domains or motifs and their function(s) is unknown. As a first step toward elucidating the function of this highly conserved family of proteins, we disrupted the TgCBAP gene by double homologous recombination. Parasites lacking TgCBAP are as sensitive to the effects of Conoidin A as wild-type parasites, demonstrating that TgCBAP is not the biologically relevant target of Conoidin A. However, ΔTgCBAP parasites are significantly shorter than wild-type parasites and have a growth defect in culture. Furthermore, TgCBAP has an unusual subcellular localization, forming small rings at the apical and basal ends of the parasite and localizing to punctate, ring-like structures around the parasite periphery. These data identify a new marker of the apical and basal subcompartments of T. gondii, reveal a potentially novel compartment along the parasite periphery, and identify TgCBAP as a determinant of parasite size that is required for a maximally efficient lytic cycle.刚地弓形虫（Toxoplasma gondii）及相关顶复门寄生虫（apicomplexan parasites）的细胞骨架具有高度极性，其顶端与基底区域由不同的蛋白质复合物构成。已知这类复合物的组分在寄生虫的形态维持、细胞分裂以及宿主细胞入侵过程中发挥关键作用。在探索刚地弓形虫入侵小分子抑制剂Conoidin A的生物学相关靶点的研究中，我们发现了一种新型细胞骨架蛋白，并将其命名为TgCBAP（保守性顶端基底外周蛋白，Conserved Basal Apical Peripheral protein）。TgCBAP的直系同源蛋白仅存在于其他顶复门寄生虫的基因组中；这类蛋白未被鉴定出可识别的结构域与基序，其具体功能尚未明确。为阐明这一高度保守蛋白质家族的功能，我们通过双同源重组技术敲除了TgCBAP基因。实验结果显示，缺失TgCBAP的寄生虫（ΔTgCBAP）与野生型寄生虫对Conoidin A的作用敏感性无显著差异，证实TgCBAP并非Conoidin A的生物学相关靶点。然而，ΔTgCBAP寄生虫的体长显著短于野生型寄生虫，且在体外培养体系中表现出生长缺陷。此外，TgCBAP具有独特的亚细胞定位模式：它在寄生虫的顶端与基底末端形成小型环状结构，同时定位于寄生虫外周的点状、环状结构区域。本研究鉴定出了刚地弓形虫顶端与基底亚区室的新型标记物，揭示了寄生虫外周一处潜在的全新亚区室，并证实TgCBAP是决定寄生虫体型大小的关键因子，对于寄生虫裂解周期的高效运转不可或缺。