Defining the shared transcriptional network underlying Toxoplasma extracellular stress and stage transition

定义弓形虫细胞外应激和阶段转变背后的共享转录网络

• 批准号: 10682134
• 负责人: Marc-Jan Gubbels
• 金额: $ 23.21万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682134
• 关键词: Acquired Immunodeficiency Syndrome; Acute; Automobile Driving; Back; Binding Sites; Biology; Carbon Dioxide; Cell Cycle; Cell Cycle Stage; Cells; Cellular Stress; Chronic; Clinical; Cluster Analysis; Communities; Congenital Abnormality; Cyst; DNA Binding; Data; Data Analyses; Data Set; Drug Targeting; Early identification; Encephalitis; Environment; Event; Exposure to; Fibroblasts; Genes; Genetic Transcription; Genotype; Human; Hypersensitivity; Immune; Immune response; Impairment; In Vitro; Individual; Infection; Interferon Type II; Knowledge; Life; Maps; Mediating; Modeling; Muscle Cells; Muscle Fibers; Neurons; Opportunistic Infections; Organ Transplantation; Parasites; Pathway interactions; Pharmaceutical Preparations; Phase; Physiological; Population; Positioning Attribute; Research; Resistance; Resources; Role; Stress; System; Time; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transcriptional Regulation; Validation; Work; biological adaptation to stress; cancer therapy; cell type; combinatorial; comparative; computational pipelines; computerized tools; experimental study; extracellular; foodborne infection; gene interaction; innovation; insight; knock-down; new therapeutic target; novel; novel therapeutic intervention; obligate intracellular parasite; programs; promoter; reference genome; response; single-cell RNA sequencing; stressor; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Infection comprises a short, acute stage (tachyzoite) followed by a dormant stage (bradyzoites in tissue cysts) for the life of the host. However, an impaired or suppressed immune response (e.g., AIDS, organ transplant, cancer treatment) can lead to reactivation of a dormant infection that was acquired years ago, leading to clinical toxoplasmosis. Interference with the tachyzoite-bradyzoite interconversion is an attractive target for novel therapeutic strategies. A key factor in stage interconversion is the ability to respond to stress: the immune response ‘stress’ maintains the bradyzoite stage, but if it wanes, the parasite defaults back to the acute stage. Reprogramming toward the bradyzoite is facilitated by the competitive interplay of about 10 transcription factors (TFs) balancing sequential, cooperative, and opposing roles. The research team recently discovered that the TF set associated with bradyzoite differentiation overlaps by 2/3 with the TFs expressed in parasites exposed to the extracellular (e.c.) environment. This provided the first glimpse of how the stress response is integrated into the transcriptional program leading to the bradyzoite. Indeed, the transcriptome of e.c. and bradyzoite parasites shares many genes, which positions the extracellular stage in between tachyzoites and bradyzoites. Here, the stress-related transcriptional program will be further dissected by extending on this observation with innovative CUT&RUN, single cell expression profiling (scRNA-seq), and the application of a novel in vitro generated myotube bradyzoite differentiation system. Excitingly, this myotube system demonstrated that spontaneous bradyzoite differentiation occurs in this cell type, but to reach and maintain a mature bradyzoite state, an exogenous, alkaline stress needs to be applied. Hence, this provides the perfect platform to dissect the contribution of stress-related transcriptional regulation to bradyzoite maturation. In short, the transcriptional network of stress will be dissected by 1. identifying the genes controlled by the 4-6 TFs shared between e.c. stress and bradyzoite conversion through CUT&RUN experiments; 2. scRNA-seq of mixed cell populations representing the transcriptional transitions of extracellular stress over the span of 6 hrs, and myotube-induced bradyzoite differentiation with and without alkaline stress. An advanced computational pipeline will be established to model the transitional programs that will permit answers to several open questions, such as: is there indeed a two-step bradyzoite differentiation pathway, and what are the genes driving these?; is the myotube seen as a stress by the parasites or not?; is there a specific stage in the cell cycle when bradyzoite differentiation occurs (a G2-like cell cycle stage and the late G1 restriction-checkpoint have been proposed)? In addition, comparative mapping of stress responses under different conditions will provide insights in how stress is integrated at the distinct parasite stages to modulate the stage-specific transcriptional responses.

概括 弓形虫是一种专性细胞内顶复门寄生虫，可引起严重的机会性感染。 目前容易诱发药物过敏，尤其是短期、感染后。 急性阶段（速殖子），随后是宿主一生的休眠阶段（组织包囊中的缓殖子）。 然而，免疫反应受损或受到抑制（例如艾滋病、器官移植、癌症治疗）可以 导致多年前获得的休眠感染重新激活，导致临床弓形虫病。 干扰速殖子-缓殖子相互转化是新型治疗策略的一个有吸引力的目标。 阶段转换的一个关键因素是应对压力的能力：免疫反应“压力”维持 缓殖子阶段，但如果它减弱，寄生虫就会默认回到急性阶段。 大约 10 个平衡顺序转录因子 (TF) 的竞争性相互作用促进了缓殖子的形成， 研究小组最近发现 TF 集与合作和对立角色相关。 缓殖子分化与暴露于细胞外（e.c.）的寄生虫中表达的 TF 重叠 2/3 这首次展示了应激反应如何整合到转录中。 事实上，e.c. 和缓殖子寄生虫的转录组有许多相似之处。 基因，将细胞外阶段定位在速殖子和缓殖子之间，这里是与应激相关的。 转录程序将通过创新的 CUT&RUN 扩展这一观察来进一步剖析， 单细胞表达谱（scRNA-seq），以及新型体外生成的肌管缓殖子的应用 令人兴奋的是，该肌管系统证明了自发的缓殖子分化。 发生在这种细胞类型中，但为了达到并维持成熟的缓殖子状态，需要外源性碱性应激 因此，这提供了一个完美的平台来剖析压力相关转录的贡献。 简而言之，应激的转录网络将被 1 所剖析。 识别由 e.c. 应激和缓殖子转化之间共享的 4-6 个 TF 控制的基因 2. 代表转录转变的混合细胞群的 scRNA-seq； 6小时内的细胞外应激，以及肌管诱导的缓殖子分化（有或没有） 将建立先进的计算管道来模拟过渡程序 将回答几个悬而未决的问题，例如：是否确实存在两步缓殖子分化 途径，驱动这些途径的基因是什么？；寄生虫是否将肌管视为应激？； 当缓殖子分化发生时，细胞周期中有一个特定的阶段（类似 G2 的细胞周期阶段和 已提出晚期 G1 限制检查点）？此外，压力反应的比较图 在不同的条件下，将提供关于压力如何在不同的寄生虫阶段整合的见解 调节阶段特异性转录反应。