Rimple Patrick A, Olafsson Einar B, Markus Benedikt M, Wang Fengrong, Augusto Leonardo, Lourido Sebastian, Carruthers Vern B
bioRxiv. 2024 Nov 27:2024.11.27.625683. doi: 10.1101/2024.11.27.625683.
The obligate intracellular parasite replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and Δ , Δ , and Δ mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that Δ and Δ mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway's potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the ingestion pathway during nutrient scarcity.
is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of .
这种专性细胞内寄生虫在一种称为寄生泡（PV）的特殊区室中进行复制。最近的研究表明，尽管它生活在寄生泡内，但它通过一种所谓的摄取途径从受感染宿主细胞的细胞质中内吞蛋白质。摄取途径由致密颗粒蛋白GRA14启动，GRA14与宿主内体分选转运复合体（ESCRT）机制结合，促使囊泡芽生进入寄生泡腔。含有蛋白质的囊泡被寄生虫内化，并被运输到植物液泡样区室（PLVAC），在那里组织蛋白酶L（CPL）降解货物，而氯喹抗性转运蛋白（CRT）将产生的肽和氨基酸输出到寄生虫细胞质中。然而，尽管摄取途径被认为是营养物质的通道，但这一假设的证据有限。我们推断，如果寄生虫利用摄取途径获取营养，那么缺乏GRA14、CPL或CRT的寄生虫应该更多地依赖生物合成途径或替代清除途径。为了探究这一点，我们在野生型（WT）寄生虫以及ΔGRA14、ΔCPL和ΔCRT突变体中进行了全基因组CRISPR筛选，以鉴定在摄取缺陷型寄生虫中更能赋予适应性的基因。我们的筛选揭示，与WT相比，在摄取突变体中更能赋予适应性的基因存在显著重叠。通路分析表明，ΔGRA14和ΔCPL突变体更多地依赖嘧啶生物合成、脂肪酸生物合成、三羧酸循环和赖氨酸降解。大量代谢组学分析显示，与WT相比，摄取突变体中的糖酵解中间产物和氨基酸水平降低，突出了该途径在宿主资源清除中的潜在作用。有趣的是，摄取突变体在氨基酸耗尽的培养基中生长时表现出更严重的生长缺陷，这表明寄生虫摄取途径在营养缺乏期间发挥了作用。
疟原虫是一种专性细胞内病原体，几乎可感染大多数温血动物的任何有核细胞。大多数情况下感染是无症状的，但免疫力较弱的人可能会患上严重疾病。为了在宿主体内进行复制，寄生虫必须有效地获取必需营养，尤其是因为它无法合成几种关键代谢物。了解疟原虫从宿主获取营养的机制对于确定潜在治疗靶点至关重要。我们的研究突出了摄取途径在维持寄生虫代谢物方面的作用，并有助于在氨基酸限制条件下寄生虫的复制。这项工作推进了我们对疟原虫代谢适应性的理解。
