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 HIV/AIDS重要条件致病菌--弓形虫的细胞周期 从根本上说，这与典型的真核生物是不同的。活跃增长中的复制 速殖子在急性感染和缓殖子内与慢性感染相关的情况下由体内 萌发过程称为内生作用。在内生的情况下，每个寄生虫的母体每只生下两个女儿 周而复始。相比之下，寄生虫的有性周期，这一阶段仅限于猫科动物的肠道，由 分裂和内多基因，通常与疟疾寄生虫和 肉孢子虫属(Sarcocystis)。这些细胞周期结构与多个 每个复制周期的子代。我们最近鉴定了一种细胞周期调节的脱泛素酶TgOTUD3A (TgGT1_258780)其靶向中断放宽了对速殖子复制的限制 内源(每个周期2个后代)同时表现出分裂和内源多基因的特征--通常 在相同的克隆液泡中。这些突变寄生虫每周期产生3，4或5个后代，以定义 多女儿表型。此外，超过70%的TgOTUD3A速殖子异位表达标志物 与缓殖子相关，并诱导通常与裂殖子相关的基因的表达， 猫科动物进入有性周期的阶段。TgOTUD3A-KO的互补失败 恢复这些表型中的任何一个，全基因组测序显示没有可信的突变 基因组水平。这表明已经发生了一种补偿性适应。对对方的检查 TgOTU家族成员发现两个亲缘关系密切的家族选择性转录上调 成员TgOTUD1B(TgGT1_237894)和TgOTUD1C(TgGT1_323200)。很可能是TgOTUD1a (TgGT1_207650)也同样上调。这表明，一个或多个细胞周期和 TgOTUD3A基因缺失的发育表型实际上可能是由于TgOTUD3A基因表达上调所致 D1TgOTU成员。为了解决这个特定问题，我们建议从功能上描述 D1TgOTU成员并确定其消融和受管制的后果 在野生型和TgOTUD3A-KO背景下过表达。通过这样做，我们希望获得对以下方面的见解 这些去泛素酶如何控制细胞周期结构的选择和关键基因的控制方面 发展转型巩固了这些基本过程之间正在形成的联系。