Sphingolipid and Mechanism of Cyst Formation by Giardia

描述（由申请人提供）：贾第鞭毛虫是一种水媒寄生虫，在发达国家和发展中国家都是导致肠道感染的原因。贾第虫病也是一种人畜共患疾病，通过受污染的水通过感染囊肿传播。囊肿在通过人体胃的过程中暴露于胃酸会引发囊肿排出，而滋养体定植的小肠中的因素则会诱导成囊或囊肿形成。囊化的标志是囊化特异性囊泡（esv）的生物发生，这些囊泡运输囊壁物质，这些物质随后与质膜融合并形成囊壁。然而，尚不清楚ESV的生物发生是如何调控的，以及如何产生活的或感染性的囊肿。我们实验室的结果表明贾第鞭毛虫表达较少的鞘脂生物合成基因，这些基因在胞制过程中受到差异调节。糖基神经酰胺转移酶-1 （glucosylceramide transferase-1, gGlcT1）是SL生物合成的重要酶之一，其过表达可导致esv增大和聚集，并改变细胞脂质稳态。另一方面，gllct1的敲低会干扰ESV的形成，从而导致隐性囊肿的产生，其生存能力降低。基于这些观察结果，我们假设gGlcT1的调控表达对于ESV的生物发生和传播疾病的感染囊肿的产生至关重要。在Specific Aim 1中，gGlcT1调节脂质稳态和ESV生物发生的机制将通过gGlcT1过表达和敲除贾第虫来研究。我们将确定gGlcT1是否与鞘脂生物合成途径的其他酶协同调节ESV的形成。参与功能性esv组装的脂质成分将通过质谱和分子/细胞方法进行鉴定。在特异性目标2中，将确定gGlcT1在调节囊肿形态、活力和感染性中的作用。gGlcT1敲低产生的隐囊的生物活性和感染性将分别在体外切除和体内动物模型中进行测试。将确定负责改变囊肿形态和活力的囊壁成分。这项拟议的研究将确定gGlcT1作为贾第鞭毛虫囊肿形成的一种新的调节因子，并可能用于开发控制贾第鞭毛虫病的新疗法。贾第鞭毛虫病每年影响全世界数百万儿童。

DESCRIPTION (provided by applicant): Giardia lamblia, a waterborne parasite, is responsible for intestinal infections in both developed and developing countries. Giardiasis, which is also a zoonotic disease, is transmitted via infective cysts through contaminated water. Exposures of cysts to gastric acid during passage through the human stomach trigger excystation, while factors in the small intestine, where trophozoites colonize, induce encystation or cyst formation. The hallmark of encystation is the biogenesis of encystation-specific vesicles (ESVs), which transport cyst-wall materials that later merge with the plasma membrane and lay down the cyst wall. However, it is not clear how ESV biogenesis is regulated and how viable or infective cysts are produced. Results from our laboratory have indicated that Giardia expresses fewer sphingolipid biosynthesis genes, which are differentially regulated during encystation. The overexpression of glucosylceramide transferase-1 (gGlcT1), one of the important enzymes of SL biosynthesis, produces enlarged and aggregated ESVs and alters cellular lipid homeostasis. The knockdown of gGlcT1, on the other hand, interferes with ESV formation that leads to the generation of cryptic cysts with reduced viability. Based on these observations, we hypothesize that the regulated expression of gGlcT1 is essential for ESV biogenesis and the production of infective cysts that transmit the disease. In Specific Aim 1, the mechanism by which gGlcT1 regulates lipid homeostasis and ESV biogenesis will be investigated using gGlcT1 overexpressed and knockdown Giardia. We will determine whether gGlcT1 coordinates with other enzymes of sphingolipid biosynthetic pathway to regulate ESV formation. The lipid components involved in assembly of functional ESVs will be identified by mass spectrometry and molecular/cellular methodologies. In Specific Aim 2, the role of gGlcT1 in regulating cyst morphology, viability, and infectivity will be determined. The biological activity and infectivity of cryptic cysts produced by gGlcT1 knockdown will be tested in in vitro excystation and in vivo animal model, respectively. The cyst- wall components that are responsible for altering cyst morphology and viability will be determined. The proposed study will identify gGlcT1 as a novel regulator of cyst formation by Giardia and could be exploited for the development of new therapies to control giardiasis, which affects millions of children worldwide each year.