Synthetic lethality screens in a nutrient sensitized Toxoplasma strain to identify novel proteins that mediate nutrient acquisition in chronic Toxoplasma infection.

细胞内寄生虫刚地弓形虫从快速分裂的速殖子阶段转变为缓慢分裂的 组织包囊缓殖子阶段是感染的慢性期的特征。弓形虫 这些半休眠的囊肿重新激活引起的脑炎(TE)仍然是发病的重要原因 和艾滋病患者的死亡率。目前的药物耐受性很差，而且不能杀死缓殖子。 各阶段。因此，迫切需要鉴定对缓殖子很重要的弓形虫蛋白。 在包囊内存活，这样就可以设计出针对这些蛋白质的新疗法。 弓形虫在被寄生虫泡(PV)膜包围的寄生虫空泡(PV)内复制 (PVM)。为了满足营养缺乏症的要求，寄主营养物质需要越过PVM屏障，然而毒素- PVM中参与营养获取的血浆蛋白在很大程度上是未知的。我们发现有两个 弓形虫蛋白GRA17和GRA23是由其致密的颗粒分泌组织分泌的。 NELL进入PV管腔，在PVM中形成毛孔，调节小分子的流量，如Nutri- Ents，穿过PVM。ΔGra17寄生虫生长缓慢，在小鼠中是无毒的，与某些 营养物质限制了ΔGra17寄生虫的生长。这个项目的基本原理是我们重新- 最近的研究表明，GRA17对包囊内缓殖子的生存也很重要，因此弓形虫-- 调节：(I)在PVM获得营养的血浆蛋白；或(Ii)参与 从PV腔到PVM的营养获取，可能决定了包囊内缓殖子的存活率。 最近，我们进行了CRISPR/Cas9介导的全基因组功能丧失筛查，并 鉴定出在营养致敏的∆Gra17中有很强适合性缺陷的弓形虫基因，但没有 在野生型寄生虫中。因为∆Gra17∆Gra23寄生虫是不能存活的，我们的工作假设是 参与GRA23向PVM运输的弓形虫基因，在其分泌到PV腔后，是 在我们的屏幕上，可能还有其他基因调节PVM的营养吸收。怎么回事- 然而，缺少的电源证实了这个屏幕上的大多数点击量。因此，在我们的第一个目标中，我们将- 来自CRISPR/CAS9功能丧失屏幕的财年命中，这些屏幕在∆Gra17中是人工致死/患病的 网站。在我们的第二个目标中，我们将识别弓形虫基因，这些基因决定了体内缓殖子的生存。 通过：(I)调节PVM的营养获取；或(Ii)影响蛋白质在- 向PVM输送营养物质。在R21完成后，我们的预期结果是 已经确定了新的弓形虫蛋白，这些蛋白对包囊内缓殖子的生存能力很重要。 这些结果预计将产生重要的积极影响，因为它们将提供新的目标 为合理开发目前尚不存在的治疗弓形虫的药物提供依据。

The intracellular parasite Toxoplasma gondii converts from fast-dividing tachyzoite stages to slowly divid- ing tissue-encysted bradyzoite stages that characterize the chronic phase of the infection. Toxoplasma encephalitis (TE) from reactivation of these semi-dormant cysts remains an important cause of morbidity and mortality in AIDS patients. Current drugs are poorly tolerated, and they do not kill the bradyzoite stages. Thus, there is a critical need to identify Toxoplasma proteins that are important for bradyzoite survival inside cysts, such that novel therapeutics can be designed to target these proteins. Toxoplasma replicates within a parasitophorous vacuole (PV) surrounded by the PV membrane (PVM). To satisfy its auxotrophic requirements, host nutrients need to cross the PVM barrier, yet Toxo- plasma proteins in the PVM involved in nutrient acquisition are largely unknown. We discovered that two Toxoplasma proteins, GRA17 and GRA23, which are secreted from its dense granule secretory orga- nelles into the PV lumen, make pores in the PVM that mediate the flux of small molecules, such as nutri- ents, across the PVM. Δgra17 parasites are slow growing and avirulent in mice, consistent with certain nutrients becoming limiting to Δgra17 parasite growth. The rationale for this project is that we have re- cently shown that GRA17 is also important for survival of bradyzoites inside cysts and therefore Toxo- plasma proteins that mediate: (i) nutrient acquisition at the PVM; or (ii) trafficking of proteins involved in nutrient acquisition from the PV lumen to the PVM, likely determine survival of bradyzoites inside cysts. Recently, we performed a CRISPR/Cas9-mediated genome-wide loss-of-function screen and identified Toxoplasma genes that have a strong fitness defect in the ‘nutrient sensitized’ ∆gra17 but not in wild-type parasites. Because ∆gra17∆gra23 parasites are not viable, our working hypothesis is that Toxoplasma genes involved in trafficking of GRA23 to the PVM, after its secretion into the PV lumen, are likely among the hits of our screen as are other genes mediating nutrient uptake at the PVM. What re- mains lacking, however, is confirming most hits from this screen. Therefore, in our first aim we will identi- fy hits from the CRISPR/Cas9 loss-of-function screens that are synthetically lethal/sick in ∆gra17 para- sites. In our second aim we will identify Toxoplasma genes that determine survival of bradyzoites inside cysts by: (i) mediating nutrient acquisition at the PVM; or (ii) affecting correct trafficking of proteins in- volved in nutrient acquisition to the PVM. At the completion of this R21, our expected outcomes are to have identified novel Toxoplasma proteins that are important for viability of bradyzoites inside cysts. These results are expected to have an important positive impact because they will provide new targets for the rational development of therapeutics against Toxoplasma bradyzoites which currently do not exist.