Extragenic adaptation to the loss of a deubiquitinase affecting the T. gondii cell cycle and development

项目摘要 HIV/AIDS的重要条件致病菌--弓形虫的细胞周期 患者，从根本上不同于典型的真核生物。复制在积极增长的 急性感染中的速殖子和与慢性感染相关的缓殖子内的速殖子通过内部 出芽过程称为endodysis。内分泌失调时，每只母寄生虫产生2个子代。 周期与此相反，寄生虫的性周期，一个阶段仅限于猫的肠道，发生的杂交， 典型地与疟疾寄生虫和寄生虫的成员相关的寄生虫和内生多殖 属Sarcocystis。这些细胞周期结构与多个细胞周期的产生相关。 每个复制周期的后代。我们最近鉴定了细胞周期调节的去泛素化酶TgOTUD 3A （TgGT1_258780），其靶向破坏放松了速殖子复制的限制， endodygenesis（每个周期2个后代）表现出雌配子体生殖和内多生殖的特征-通常 在同一个克隆液泡中。这些突变体寄生虫每个周期产生3、4或5个后代，以确定 多子代表型。此外，超过70%的TgOTUD 3A速殖子异位表达标记 与缓殖子相关并诱导通常与裂殖子相关的基因的表达， 与进入猫的性周期有关的阶段。TgOTUD 3A-KO的互补未能 恢复这些表型中的任何一种，全基因组测序显示， 基因组水平。这表明发生了补偿性适应。检查其他 TgOTU家族成员揭示了两个密切相关的家族的选择性转录上调 成员，TgOTUD 1B（TgGT1_237894）和TgOTUD 1C（TgGT1_323200）。TgOTUD 1A可能 （TgGT1_207650）类似地上调。这表明一个或多个细胞周期和 归因于TgOTUD 3A缺失的发育表型实际上可能是由于TgOTUD 3A的上调。 进化枝D1 TgOTU成员。为了解决这个具体问题，我们建议在功能上描述 进化枝D1 TgOTU成员，并建立它们的消融和调节的结果。 在野生型和TgOTUD 3A-KO背景中的过表达。在这样做的过程中，我们希望了解 这些去泛素化酶如何支配细胞周期结构的选择和关键的 发展转型巩固了这些基本进程之间正在出现的联系。

Project Summary The cell cycle of protozoan parasite Toxoplasma gondii, an important opportunistic pathogen in HIV-AIDS patients, is fundamentally distinct from that of typical eukaryotes. Replication in the actively growing tachyzoites in acute infection and within bradyzoites associated with chronic infection occurs by an internal budding process termed endodyogeny. With endodyogeny, each mother parasite produces 2 daughters per cycle. In contrast, the sexual cycle of the parasite, a stage restricted to the feline gut, occurs by a hybrid of schizogony and endopolygeny which are typically associated with the malaria parasite and members of the genus Sarcocystis respectively. These cell cycle architectures are associated with the generation of multiple progeny per replicative cycle. We recently characterized a cell cycle regulated deubiquitnase TgOTUD3A (TgGT1_258780) the targeted disruption of which relaxed the restriction of tachyzoites replication to endodyogeny (2 progeny per cycle) to exhibit characteristics of both schizogony and endopolygeny-often within the same clonal vacuole. These mutant parasites generate 3,4 or 5 progeny per cycle to define the multi-daughter phenotype. In addition, over 70% of TgOTUD3A tachyzoites ectopically express markers associated with bradyzoites and induce the expression of genes typically associated with merozoties, the stage associated with entry into the sexual cycle in cats. Complementation of the TgOTUD3A-KO failed to restore any of these phenotypes, with whole genome sequencing revealing no credible mutations at the genomic level. This suggested that a compensatory adaptation had occurred. Examination of the other TgOTU family members revealed a selective transcriptional upregulation of two closely related family member, TgOTUD1B (TgGT1_237894) and TgOTUD1C (TgGT1_323200). It is likely that TgOTUD1A (TgGT1_207650) is similarly upregulated. This presents the possibility that one or more of the cell cycle and developmental phenotypes attributed to the loss of TgOTUD3A may in fact be due to the upregulation of the Clade D1 TgOTU members. To address this specific question we propose to functionally characterize the Clade D1 TgOTU members and establish the consequence of both their ablation and regulated overexpression in the wild type and TgOTUD3A-KO background. In doing so we expect to gain insights into how these deubiquitinases govern the selection of cell cycle architecture and control aspects of key developmental transitions cementing an emerging association between these fundamental processes.