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

对影响弓形虫细胞周期和发育的去泛素酶损失的外源适应

• 批准号: 9914617
• 负责人: ANTHONY P. SINAI
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-20 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9914617
• 关键词: Ablation; Acquired Immunodeficiency Syndrome; Address; Affect; Architecture; Biochemical; CRISPR screen; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Characteristics; Chronic Phase; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Cytokinesis; Daughter; Development; Dissection; Dolichos; Ectopic Expression; Enzyme Kinetics; Epitopes; Eukaryota; Exhibits; Family; Family Felidae; Family member; Felis catus; Gap Junctions; Gene Expression; Generations; Genes; Genetic Transcription; Genomics; Growth; HIV; Hybrids; In Situ; Individual; Infection; Investigation; Lectin; Light; Link; Malaria; Mediating; Metabolism; Mitotic; Morphology; Mothers; Mutation; Nuclear; Organism; Parasites; Patients; Phenotype; Polyploidy; Process; Production; Quantitative Reverse Transcriptase PCR; Recombinants; Reporting; Role; Sarcocystis; Substrate Specificity; Surface; Toxoplasma; Toxoplasma gondii; Ubiquitin; Up-Regulation; Vacuole; Work; acute infection; asexual; chronic infection; fitness; genome sequencing; genome-wide; head-to-head comparison; insight; member; mutant; ovarian neoplasm; overexpression; pathogen; promoter; scaffold; whole genome

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.

项目概要 原生动物寄生虫弓形虫的细胞周期，一种艾滋病毒/艾滋病的重要机会病原体 患者，从根本上不同于典型的真核生物。积极成长中的复制 急性感染中的速殖子和与慢性感染相关的缓殖子是通过内部发生的 萌芽过程称为内生作用。通过内生作用，每个母寄生虫产生 2 个女儿 循环。相比之下，寄生虫的性周期（仅限于猫科动物肠道的一个阶段）是由以下因素的混合体发生的： 分裂生殖和内多生殖通常与疟原虫和疟原虫成员有关 分别为肉孢子虫属。这些细胞周期结构与多种细胞周期的产生相关。 每个复制周期的后代。我们最近表征了细胞周期调节的去泛素酶 TgOTUD3A （TgGT1_258780）有针对性的破坏，放宽了速殖子复制的限制 内生生殖（每个周期 2 个子代）通常表现出分裂生殖和内多生殖的特征 在同一个克隆液泡内。这些突变寄生虫每个周期产生 3,4 或 5 个后代，以定义 多女儿表型。此外，超过 70% 的 TgOTUD3A 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， 与猫进入性周期相关的阶段。 TgOTUD3A-KO 的互补未能 恢复任何这些表型，全基因组测序显示在这些表型上没有可信的突变 基因组水平。这表明补偿性适应已经发生。对方的检查 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，TgOTUD1B (TgGT1_237894) 和 TgOTUD1C (TgGT1_323200)。 TgOTUD1A 很可能 (TgGT1_207650) 类似地上调。这提出了一种或多种细胞周期和 归因于 TgOTUD3A 缺失的发育表型实际上可能是由于 D1 TgOTU 进化枝成员。为了解决这个具体问题，我们建议从功能上表征 进化枝 D1 TgOTU 成员并确定其消融和调节的结果 在野生型和 TgOTUD3A-KO 背景下过度表达。在此过程中，我们希望深入了解 这些去泛素酶如何控制细胞周期结构的选择并控制关键的方面 发展转型巩固了这些基本过程之间的新兴联系。