Regulation of latent stage differentiation through central carbon metabolism and ubiquitination

项目总结 弓形虫潜伏感染约30%的人口，对急性期耐药 治疗。潜伏的弓形虫可作为免疫低下患者急性期重新激活的蓄水池 在其他健康的个体中，可导致渐进性失明。因此，潜伏期是 临床上很重要，但目前无法治愈。 耐药可能是由于潜伏期新陈代谢的变化，但这些仍然是一个完整的 神秘感。此外，潜伏的弓形虫在感染其体内时会暴露在不同的营养环境中。 目标组织类型-肌肉和中枢神经系统(CNS)-以及这对弓形虫的影响 延迟不被理解。我们已经发现，模拟肌肉和中枢神经系统的碳源的变化会触发 弓形虫分化为潜伏形态。然后我们进行了全基因组CRISPR筛查，并确定了 选择分化所需的弓形虫碳分解代谢酶，并进一步鉴定出 泛素化在代谢适应和分化为潜在形式中的作用。 这些背景研究为全面了解弓形虫奠定了基础 潜伏期内的新陈代谢，并了解新陈代谢重组如何通过 泛素化调控。这项计划的具体目标是： 目的1：明确潜伏弓形虫分化和持续所需的代谢途径。 我们将确定利用碳所需的特定酶(在CRISPR筛查中确定)的作用 模拟大脑和肌肉的来源及其对体外和体内分化的影响。这将决定 潜伏期和急性期的新陈代谢有何不同，以及这与环境感知和 差异化。 目的2：确定泛素化在调节分化为潜伏型中的作用。定义 E3泛素连接酶复合体的组成、生化活性和结构 分化为潜在的形式，从而决定泛素化如何调节分化。 目标3：确定泛素化如何调节新陈代谢和分化为潜在形式。我们有 确定E3泛素化机制的可能底物，并将确定它们在调节两者中的功能 新陈代谢和分化。

Project summary Toxoplasma latently infects approximately 30% of the human population and is refractory to acute stage therapies. Latent Toxoplasma can act as a reservoir for acute stage reactivation in immunocompromised individuals and can cause progressive blindness in otherwise healthy individuals. Thus, latent stages are clinically important, but currently untreatable. Drug resistance is likely due to changes in the metabolism of latent stages, but what these are remain a complete mystery. Furthermore, latent Toxoplasma is exposed to different nutritional environments when infecting its target tissue types – muscle and the central nervous system (CNS) – and the impact this has on Toxoplasma latency is not understood. We have found that changes in carbon sources, that mimic muscle and CNS trigger Toxoplasma differentiation into latent forms. We then performed a whole-genome CRISPR screen and identified select Toxoplasma carbon catabolic enzymes that are required for differentiation and furthermore, identified a role for ubiquitination in metabolic adaptation and differentiation into latent forms. These background studies form the basis for developing a comprehensive understanding of Toxoplasma metabolism during latency and understanding how metabolic rewiring is linked to differentiation through regulation by ubiquitination. The specific aims of this program are to: Aim 1: Define the metabolic pathways required for differentiation and persistence of latent Toxoplasma. We will determine the role of select enzymes (identified in CRISPR screen) required for utilization of carbon sources that mimic brain and muscle and their impact on differentiation in vitro and in vivo. This will determine how metabolism of latent and acute stages differs and how this is linked to environmental sensing and differentiation. Aim 2: Determine the role of ubiquitination in the regulation of differentiation into latent forms. Define the composition, biochemical activity and structure of an E3 ubiquitin ligase complex that is important for differentiation into latent forms, thus determining how ubiquitination regulates differentiation. Aim 3: Define how ubiquitination regulates metabolism and differentiation into latent forms. We have identified likely substrates of the E3 ubiquitination machinery and will define their function in regulation of both metabolism and differentiation.