Deciphering the composite S-phase in Toxoplasma gondii

解读弓形虫复合 S 期

• 批准号: 10744528
• 负责人: Elena Suvorova
• 金额: $ 22.5万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-16 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10744528
• 关键词: Affect; Alveolar; Antiparasitic Agents; Apicomplexa; Biology; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cell division; Characteristics; Chromosome Segregation; Closure by clamp; Cryptosporidiosis; Cyclin-Dependent Kinases; Cytokinesis; DNA; DNA biosynthesis; Daughter; Disease; Drug Targeting; Drug resistance; Engineering; Ensure; Eukaryota; Experimental Designs; Focus Groups; Future; Genetic Materials; Human; Immunocompromised Host; Infection; Knowledge; Malaria; Mitosis; Modeling; Molecular; Nuclear; Organism; Parasite resistance; Parasites; Phase; Phosphotransferases; Plasmodium; Population; Positioning Attribute; Process; Proteins; Regulation; Running; S phase; Severity of illness; Surface; Testing; Therapeutic; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Ubiquitination; Variant; asexual; burden of illness; chronic infection; controlled release; design; druggable target; experimental study; human disease; novel; nuclear division; opportunistic pathogen; pilot test; programs; segregation; sensor; temporal measurement; tool

Abstract Apicomplexa parasites contribute significantly to human disease burden, including ~1/3 of human populations permanently infected with Toxoplasma gondii. Existing treatments are limited and often toxic to the most affected population of immunocompromised patients. We need a profound knowledge of parasite biology to develop efficient anti-parasitic drugs. Our group focuses on the studies of cell cycle mechanisms that are central to parasite survival and offer a wealth of druggable targets. The cell cycle program orchestrates cell division and ensures the inheritance of the genetic material. Apicomplexan cell cycles are strikingly different from the cell cycles of their hosts. Although T. gondii tachyzoite divides by endodyogeny that resembles a binary division of the conventional eukaryotes, there are substantial differences in cell cycle organization and regulation. It includes the atypical S-phase of Toxoplasma endodyogeny, which is a primary focus of our study. The need for appropriate tools to examine the intricacy of the apicomplexan cell cycle and unconventional regulators significantly impedes the related studies. To fill a major gap in our knowledge of the essential biology of apicomplexan parasites and boost the Toxoplasma cell cycle studies, we engineered a new Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probe. In the Aim 1 experiments, we will test the hypothesis that Toxoplasma endodyogenic cell cycle includes a composite S/M/C phase that runs for nearly half of the division cycle. Using our new ToxoFUCCI probes, we will determine how the intertwined S/M/C phase is organized. In Aim 2, we will determine the mechanism of the S-phase regulation. Designed experiments will test the hypothesis that, contrary to conventional S-phase cyclin dependent kinase (Cdk), controlled release of the sequestrated Cdk-related kinase TgCrk5 regulates DNA replication in the tachyzoites. Using the conditional expression model of TgCrk5, we will determine the functions of the sequestered and the nuclear TgCrk5 and identify the TgCrk5 substrates. Given that T. gondii lacks conventional S-Cdk substrates, we expect to discover a novel TgCrk5 network. The project will advance our knowledge of the fundamental process of parasite survival and have a high potential to discover future efficient drug targets.

摘要 顶复门寄生虫对人类疾病负担有重大贡献，包括约1/3的人口 永久感染弓形虫现有的治疗方法是有限的，而且往往对受影响最严重的人有毒 免疫功能低下的患者群体。我们需要对寄生虫生物学有深刻的了解 有效的抗寄生虫药物。我们的小组专注于细胞周期机制的研究， 寄生虫的生存，并提供了丰富的药物靶点。细胞周期程序协调细胞分裂， 确保遗传物质的遗传。顶复门的细胞周期与 它们的宿主的周期。虽然T.弓形虫速殖子通过内胚层分裂，类似于 与常规的真核生物相比，在细胞周期的组织和调控方面存在着实质性的差异。它包括 弓形虫内膜炎的非典型S期，这是我们研究的主要焦点。需要 适当的工具来检查复杂的顶复体细胞周期和非常规调节剂 严重阻碍了相关研究。为了填补我们对人类基本生物学知识的一个主要空白， apicomplexan寄生虫和促进弓形虫细胞周期的研究，我们设计了一种新的荧光 基于泛素化的细胞周期指示剂（FUCCI）探针。 在目的1实验中，我们将检验弓形虫内生细胞周期包括一个周期的假设， 复合S/M/C阶段，运行近一半的分裂周期。使用我们的新型ToxoFUCCI探针， 将决定如何组织S/M/C交织阶段。在目标2中，我们将确定 S相调节。设计的实验将测试的假设，相反，传统的S-阶段 细胞周期蛋白依赖性激酶（Cdk），螯合的Cdk相关激酶TgCrk 5的受控释放调节DNA 在速殖子中复制使用TgCrk 5的条件表达式模型，我们将确定函数 的螯合和核TgCrk 5，并确定TgCrk 5底物。鉴于T.弓形虫缺乏 传统的S-Cdk衬底，我们希望发现一个新的TgCrk 5网络。该项目将推动我们的 寄生虫生存的基本过程的知识，并有很高的潜力，发现未来的有效 药物靶点