Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
PLoS Pathog. 2011 Dec;7(12):e1002410. doi: 10.1371/journal.ppat.1002410. Epub 2011 Dec 8.
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.
几种在胆固醇合成、调节、运输和信号转导中起关键作用的蛋白质通过共享进化保守的“固醇感应结构域”（SSD）而结合在一起。细胞内寄生虫弓形虫至少有一种编码含有经典 SSD 的蛋白质的基因。我们研究了这种蛋白质的作用，以提供有关寄生虫脂质调节机制的信息。该蛋白质序列预测了一种未表征的尼曼-匹克，C1 型相关蛋白（NPC1），与人类 NPC1 具有显著的同一性，并且包含许多与人类 NPC 疾病相关的残基。我们将这种 NPC1 相关蛋白命名为 TgNCR1。哺乳动物 NPC1 定位于内体溶酶体，并促进固醇和鞘脂在这些细胞器的膜之间的运动。NPC1 中的致病突变导致这些脂质在内体溶酶体中的超载。然而，TgNCR1 缺乏内体靶向信号，并且定位于弓形虫质膜下的扁平小泡。当在哺乳动物 NPC1 突变细胞中表达并正确靶向内体溶酶体时，TgNCR1 可恢复这些细胞器中胆固醇和 GM1 的清除。为了阐明 TgNCR1 在寄生虫中的作用，我们通过基因敲除破坏了 NCR1；突变寄生虫仍然存活。对 ΔNCR1 株的定量脂质组学分析显示胆固醇水平正常，但几种胆甾醇酯、神经酰胺和鞘磷脂的积累增加。ΔNCR1 寄生虫还具有丰富的储存脂质体和源自其寄生空泡的长膜管状结构。有趣的是，这些突变体可以以高频率从单个母细胞产生多个子细胞，从而允许在体外快速复制，并且它们在小鼠中的毒力比亲本株略高。这些数据表明，ΔNCR1 株已经失去了控制几种脂质细胞内水平的能力，这随后导致脂质储存、膜生物合成和寄生虫分裂的刺激。基于这些观察结果，我们认为 TgNCR1 在弓形虫的脂质动态平衡中发挥作用。
