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

对营养敏感的弓形虫菌株进行合成致死性筛选，以鉴定介导慢性弓形虫感染中营养获取的新蛋白质。

基本信息

批准号：
10097993
负责人：
JEROEN SAEIJ
金额：
$ 19.63万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-04 至 2023-01-31
项目状态：
已结题

项目摘要

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.

细胞内寄生虫弓形虫从快速分裂的速殖子阶段转变为缓慢分裂的阶段。组织包囊的缓殖子阶段是感染慢性阶段的特征。这些半休眠囊肿重新激活引起的脑炎（TE）仍然是发病的一个重要原因目前的药物耐受性差，并且不能杀死缓殖子。因此，迫切需要鉴定对缓殖子很重要的弓形虫蛋白。在囊肿内存活，这样就可以设计出针对这些蛋白质的新疗法。弓形虫在被 PV 膜包围的寄生液泡 (PV) 内复制 (PVM)，为了满足其营养缺陷的需求，宿主营养物质需要穿过 PVM 屏障，但 Toxo- PVM 中参与营养获取的血浆蛋白在很大程度上是未知的。弓形虫蛋白 GRA17 和 GRA23，由其致密颗粒分泌器官分泌内尔进入 PV 腔，在 PVM 中形成孔，介导小分子的通量，例如营养物质 Δgra17 寄生虫在小鼠体内生长缓慢且无毒，这与某些情况一致。营养物质限制了 Δgra17 寄生虫的生长。研究表明，GRA17 对于包囊内缓殖子的存活也很重要，因此 Toxo- 血浆蛋白介导：(i) PVM 的营养获取；或 (ii) 参与蛋白质的运输；从 PV 腔到 PVM 的营养获取可能决定包囊内缓殖子的存活。最近，我们进行了 CRISPR/Cas9 介导的全基因组功能丧失筛选，并确定了在“营养敏感”Δgra17 中具有强烈适应性缺陷的弓形虫基因，但没有由于 Δgra17Δgra23 寄生虫无法存活，我们的工作假设是弓形虫基因参与将 GRA23 分泌到 PV 管腔后转运至 PVM，可能是我们屏幕上的热门内容，还有其他介导 PVM 营养吸收的基因。然而，主要缺乏的是确认此屏幕上的大多数点击，因此，在我们的第一个目标中，我们将确定-。 fy 来自 CRISPR/Cas9 功能丧失筛选的命中，在 Δgra17 para- 中是综合致死/患病的在我们的第二个目标中，我们将识别决定体内缓殖子存活的弓形虫基因。包囊通过：(i) 介导 PVM 的营养获取；或 (ii) 影响蛋白质的正确运输- 参与 PVM 的营养获取完成 R21 后，我们的预期结果是：已经鉴定出新的弓形虫蛋白，这些蛋白对于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合理开发目前尚不存在的弓形虫缓殖子疗法。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma.

DOI：
10.1371/journal.ppat.1011543
发表时间：
2023-07
期刊：
PLOS PATHOGENS
影响因子：
6.7
作者：
Paredes-Santos, Tatiana C.;Bitew, Mebratu A.;Swale, Christopher;Rodriguez, Felipe;Krishnamurthy, Shruthi;Wang, Yifan;Maru, Parag;Sangare, Lamba Omar;Saeij, Jeroen P. J.
通讯作者：
Saeij, Jeroen P. J.

CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells.

DOI：
10.1128/mbio.00060-23
发表时间：
2023-04-25
期刊：
MBIO
影响因子：
6.4
作者：
Krishnamurthy, Shruthi;Maru, Parag;Wang, Yifan;Bitew, Mebratu A.;Mukhopadhyay, Debanjan;Yamaryo-Botte, Yoshiki;Paredes-Santos, Tatiana C.;Sangare, Lamba O.;Swale, Christopher;Botte, Cyrille Y.;Saeij, Jeroen P. J.
通讯作者：
Saeij, Jeroen P. J.