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。

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10097993
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Brain; CRISPR/Cas technology; Cessation of life; Chronic; Chronic Phase; Cyst; Cytoplasm; Cytoplasmic Protein; Data; Defect; Development; Diffusion; Drug Targeting; Genes; Goals; Gold; Growth; Highly Active Antiretroviral Therapy; Infection; Ingestion; Mediating; Membrane; Metabolism; Morbidity - disease rate; Mus; Nutrient; Organelles; Outcome; Parasites; Patients; Permeability; Pharmaceutical Preparations; Play; Process; Proteins; Research; Role; Secretory Vesicles; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Underrepresented Minority; Vacuole; Vesicle; base; design; fitness; genome-wide; health care availability; inhibitor/antagonist; loss of function; mortality; neuropathology; new therapeutic target; non-compliance; novel; novel therapeutics; prevent; protein transport; small molecule; therapeutic development; toxoplasmic encephalitis; trafficking; uptake

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弓形虫从快速分散的速度阶段转化为缓慢分裂 为特征的感染的慢性期表征组织 - 屈肌的胸膜阶段。弓形虫 这些半休眠囊肿的重新激活的脑炎（TE）仍然是发病率的重要原因 和艾滋病患者的死亡率。目前的药物的耐受性不佳，并且不会杀死Bradyzoite 阶段。这是肯定需要识别对牛adzoite很重要的毒素蛋白 囊肿内的生存，可以设计出新的疗法来靶向这些蛋白质。 弓形虫在被PV膜包围的寄生液泡（PV）内复制 （PVM）。为了满足其可营养的要求，宿主营养素需要穿越PVM屏障，尚未有毒 参与营养获得的PVM中的血浆蛋白在很大程度上未知。我们发现两个 弓形虫蛋白，GRA17和GRA23，它们从其密集的颗粒秘书Orga-分泌 nelles进入PV腔，在PVM中制成孔，以介导小分子的通量，例如营养 Ent，穿过PVM。 Δgra17寄生虫在小鼠中的生长缓慢且无动于衷，与某些 营养成为ΔGra17寄生虫生长的限制。该项目的理由是我们有 曲目表明，GRA17对于囊肿内的胸肌的存活也很重要，因此有毒剂 - 介导的血浆蛋白：（i）PVM的营养获取；或（ii）涉及蛋白质的运输 从PV Lumen到PVM的养分获取可能确定囊肿内的Bradyzoites的存活。 最近，我们进行了CRISPR/CAS9介导的全基因组丧失屏幕， 鉴定出在“营养敏感” ∆Gra17中具有很强适应性缺陷的弓形虫基因，但不是 在野生型寄生虫中。因为∆gra17Δgra23寄生虫不可行，所以我们的工作假设是 分泌到PV管腔后，毒品基因与一起运输GRA23的毒品基因是 在我们的屏幕命中率中可能与其他介导PVM营养吸收的基因一样。什么 但是，缺乏电源正在证实此屏幕中的大多数命中。因此，在我们的第一个目标中，我们将确定 - FY命中来自CRISPR/CAS9功能丧失屏幕，这些屏幕在Δgra17para中是致命的/病态的。 站点。在我们的第二个目标中，我们将确定确定Bradyzoites内部存活的毒品基因 囊肿作者：（i）介导PVM的养分获取；或（ii）影响蛋白质的正确运输 - 将营养成分征收到PVM。在此R21完成时，我们的预期结果是 已经确定了新型的毒素蛋白，这些蛋白对于囊肿内的胸肌的生存力很重要。 预计这些结果将产生重要的积极影响，因为它们将提供新的目标 为了合理地开发针对目前不存在的弓形虫的毒品。

项目成果

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.