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.

项目摘要 原生动物寄生虫弓形虫的细胞周期，这是HIV-AID中重要的机会性病原体 患者从根本上与典型的真核生物不同。积极成长的复制 急性感染和与慢性感染相关的Bradyzoites中的速二鼠发生在内部发生 发芽过程称为内部发展。每个母亲寄生虫都有内部发育，每个寄生虫会产生2个女儿 循环。相比之下，寄生虫的性周期（仅限于猫肠）的阶段是由 精神分裂症和内部人物通常与疟疾寄生虫和成员有关 肉眼属属。这些细胞周期架构与多重生成有关 每个复制周期后代。我们最近表征了细胞周期调节的去泛素化酶TGOTUD3A （TGGT1_258780）目标破坏放宽了tachyzoites复制的限制 内部发育（每个周期2个后代），表现出精神分裂和大都会的特征 在同一克隆液泡中。这些突变的寄生虫每个周期产生3,4或5个后代，以定义 多笑表型。此外，超过70％的tgotud3a速二鼠异位表达标记 与Bradyzoites相关，并诱导基因的表达，通常与Merozoties相关， 与进入猫的性周期有关的阶段。 tgotud3a-ko的互补未能 恢复这些表型中的任何一个，整个基因组测序揭示了没有可靠的突变 基因组水平。这表明发生了补偿性适应。审查对方 TGOTU家庭成员揭示了两个密切相关家庭的选择性转录上调 成员，TGOTUD1B（TGGT1_237894）和TGOTUD1C（TGGT1_323200）。 tgotud1a可能 （TGGT1_207650）同样上调。这表明了一个或多个细胞周期和多个的可能性 归因于tgotud3a丢失的发育表型实际上可能是由于上调 进化枝D1 TGOTU成员。为了解决这个特定问题，我们建议在功能上表征 D1 TGOTU成员以及建立其消融和受监管的后果 野生型和TGOTUD3A-KO背景中的过表达。在这样做的过程中，我们希望能够了解 这些去泛素酶如何控制细胞周期结构的选择和关键的控制方面 发展过渡巩固了这些基本过程之间的新兴关联。