Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma.
Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma.
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DOI：
10.1371/journal.ppat.1011543
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发表时间：
2023-07
期刊：
影响因子：
6.7
通讯作者：
Saeij, Jeroen P. J.
中科院分区：
文献类型：
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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.
弓形虫是一种寄生虫，在一个被称为寄生虫空泡（PV）的专门隔室中复制，该隔室被PV膜（PVM）包围。为了获得必需的营养物质，弓形虫必须通过PVM转运分子，这是一个由分泌的寄生虫蛋白GRA17和GRA23介导的过程。这些蛋白质在PVM中形成孔，小分子可以通过这些孔扩散进出PV。GRA17和GRA23是合成致死的，这表明至少一种孔类型是寄生虫生存所必需的。在"营养物致敏的" Δ gra17菌株中，其他弓形虫基因很可能成为必需的，因为它们介导从宿主获得营养物或参与GRA23向PVM的运输。为了鉴定这些基因，在野生型和Δ gra17寄生虫中进行了全基因组功能丧失筛选，其鉴定了与GRA17合成致病/致死的多个基因。这些基因中有几个参与了GRA（包括GRA17/GRA23）到PVM的正确定位。预测最高命中之一GRA72在PVM上形成孔，并且其缺失导致形成扩大的"气泡空泡"，同时PVM小分子渗透性降低，类似于先前在Δ gra17寄生虫中观察到的情况。此外，Δ gra72寄生虫在小鼠中的体外生长和毒力降低。这些发现表明，在没有GRA17的情况下，其他基因变得必不可少，可能是因为它们在GRA23（和其他GRA）的适当定位中发挥作用，或者因为它们决定了PVM处宿主来源的营养物质的获取。病原体存在于宿主细胞内的膜结合液泡中，需要克服液泡膜屏障以获得宿主营养。空泡寄生虫弓形虫使用两种蛋白质GRA17和GRA23在空泡膜上形成孔，这是小宿主分子（如营养物质）穿过膜扩散所必需的。GRA17和GRA23从寄生虫的致密颗粒分泌细胞器中被外吞到液泡腔中。然而，随后确定其适当定位到液泡膜的确切机制尚不清楚。在缺失GRA17基因（Δ gra17）的寄生虫中，GRA23成为寄生虫存活的关键因素。我们利用这种脆弱性进行了一次筛选，确定了哪些寄生虫基因在Δ gra17中是必需的，而在野生型寄生虫中不是。该筛选鉴定了多种弓形虫基因，其编码多种致密颗粒蛋白（包括GRA17和GRA23）正确定位所需的蛋白质。由于致密颗粒蛋白可以介导宿主-寄生虫相互作用，因此，致密颗粒蛋白的正确定位所需的寄生虫蛋白对于寄生虫适应性至关重要。我们的筛选还鉴定了一种致密颗粒蛋白，其具有在液泡膜中形成孔的预测能力。这些气孔可能在促进宿主营养物质的获取方面发挥作用。
Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the ‘nutrient sensitized’ Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged “bubble vacuoles” with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM. Pathogens that reside in a membrane-bound vacuole inside host cells need to overcome the vacuole membrane barrier to acquire host nutrients. The vacuolar parasite Toxoplasma gondii makes pores in the vacuole membrane using two proteins, GRA17 and GRA23, that are required for the diffusion of small host molecules, such as nutrients, across the membrane. GRA17 and GRA23 are exocytosed from the parasite’s dense granule secretory organelles into the vacuolar lumen. However, the exact mechanism that subsequently determines their proper localization into the vacuole membrane is not known. In parasites that are missing the GRA17 gene (Δgra17), GRA23 becomes a critical factor for parasite survival. We exploited this vulnerability by conducting a screen that determined what parasite genes are essential in Δgra17 but not in wild-type parasites. This screen identified multiple Toxoplasma genes that encode for proteins that are required for the correct localization of multiple dense granule proteins, including GRA17 and GRA23. Because dense granule proteins can mediate host-parasite interactions, the parasite proteins that are needed for the correct localization of dense granule proteins are critical for parasite fitness. Our screen also identified a dense granule protein with the predicted ability to form pores in the vacuole membrane. These pores are likely to play a role in facilitating the acquisition of host nutrients.
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