Deficiency of a Niemann-Pick, Type C1-related Protein in Toxoplasma Is Associated with Multiple Lipidoses and Increased Pathogenicity

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved ‘sterol-sensing domain’ (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.多种在胆固醇合成、调控、转运及信号传导中发挥关键作用的蛋白质，均拥有系统发育保守的固醇感知结构域（sterol-sensing domain, SSD）。细胞内寄生虫弓形虫（Toxoplasma）至少拥有一个编码含典型SSD的蛋白质的基因。本研究对该蛋白质的功能展开探究，以解析该寄生虫的脂质调控机制。该蛋白质的序列预测显示其为未被表征的尼曼-皮克C1型相关蛋白（Niemann-Pick, type C1-related protein, NPC1），与人类NPC1具有显著同源性，且包含多个与人类NPC疾病相关的残基，我们将其命名为TgNCR1。哺乳动物NPC1定位于内体溶酶体（endo-lysosomes），可促进固醇与鞘脂跨这些细胞器膜转运。NPC1的致病错义突变会导致这些脂质在内体溶酶体中过度蓄积。但TgNCR1缺乏内体靶向信号，定位于弓形虫质膜下方的扁平囊泡中。当在哺乳动物NPC1突变细胞中表达并正确靶向至内体溶酶体时，TgNCR1可恢复这些细胞器中胆固醇与神经节苷脂GM1（GM1）的清除能力。为明确TgNCR1在寄生虫中的功能，我们通过遗传学手段敲除了NCR1基因；敲除后的突变寄生虫仍可存活。对ΔNCR1株的定量脂质组学分析显示，其胆固醇水平正常，但多种胆固醇酯、鞘磷脂及神经酰胺物种出现过度蓄积。ΔNCR1寄生虫还表现出丰富的贮存脂滴，以及源自纳虫空泡（parasitophorous vacuoles）的长膜管状结构。有趣的是，这些突变体可高频实现单个母细胞产生多个子代的分裂方式，使其在体外能够快速增殖，且在小鼠体内的毒力略高于亲本菌株。上述数据表明，ΔNCR1株丧失了对多种脂质细胞内水平的调控能力，进而刺激了脂质贮存、膜生物合成及寄生虫分裂。基于上述观察，我们认为TgNCR1在弓形虫的脂质稳态（lipid homeostasis）调控中发挥重要作用。