Sphingolipids as Potential Targets for Anti-Giardial Therapy

蓝氏贾第鞭毛虫是一种非侵入性寄生虫，可引起人类的水传播感染。这 有鞭毛的原生动物以两种形式存在，即，滋养体和包囊。形态 从包囊到滋养体的转化（脱囊）发生在胃中，而 小肠触发滋养体向包囊的转化（包囊形成）。这个项目的主要目标是 建议是阐明鞘脂在调节成囊-脱囊周期中的新作用， 贾第虫众所周知，鞘脂在信号传导、分化和凋亡中起着重要作用 在所有真核细胞中。然而，贾第虫合成鞘脂的能力有限， 鞘脂的外源性供应，用于能量产生和膜生物合成。我们的研究 这表明贾第虫使用肌动蛋白/网格蛋白依赖性途径来输入和靶向神经酰胺， 鞘脂的前体，到达ER/核周膜。分子分析显示，只有两个 鞘脂代谢基因在滋养体和包囊贾第虫中差异表达。SPT-2 （丝氨酸棕榈酰转移酶-2）mRNA主要在滋养体中表达，而GlcT-1 （神经酰胺-葡糖基转移酶）转录物主要在包囊细胞中表达。的结果 SPT-2基因在滋养体中的上调，我们推测合成的3-酮-神经鞘氨醇 由SPT和所需的肌动蛋白聚合和内吞作用，增加内吞交通， 滋养体然而，对于囊壁生物合成，我们假设外源性神经酰胺被吸收， 并用作支架组装复杂的含糖神经酰胺/鞘脂 在GlcT-1酶（由GlcT-1基因编码）的帮助下。为了验证这些假设，我们提出 以下目标，即，目的-1：确定是否存在SPT-2和GlcT-1基因和酶， 在脱囊和包囊贾第虫中差异调节;目的-2：确定转录后是否 SPT-2和GlcT-1基因的沉默将中断脱囊-成囊循环;和Aim-3： 确定SPT-2和GlcT-1 mRNA或基因产物是否对神经酰胺内吞作用至关重要 和代谢，以及包囊特异性囊泡和包囊壁的合成。 包囊细胞 这些研究将产生关于鞘脂在调节神经元细胞凋亡中的作用的有价值的信息。 心血管生命周期，并将奠定基础，为未来的努力，开发新的疗法，针对这一点 水媒病原体

Giardia lamblia is a non-invasive parasite that can cause waterborne infection in humans. This flagellated protozoan exists in two forms, i.e., the trophozoite and the cyst. The morphological transformation from cyst to trophozoite (excystation) takes place in the stomach, while factors in the small intestine trigger the transformation from trophozoite to cyst (encystation). The major goal of this proposal is to elucidate the novel role of sphingolipids in regulating the encystation-excystation cycle of Giardia. It is well known that sphingolipids play an important role in signaling, differentiation and apoptosis in all eukaryotic cells. However, Giardia has a limited ability to synthesize sphingolipids and depends on an exogenous supply of sphingolipids for energy production and membrane biosynthesis. Our studies indicate that Giardia uses actin/clathrin-dependent pathways to import and target ceramide, the major precursor of sphingolipids, to the ER/perinuclear membranes. Molecular analysis revealed that only two sphingolipid metabolic genes are differentially expressed in trophozoites and encysting Giardia. SPT-2 (serine-palmitoyl transferase-2) mRNA is expressed predominantly in trophozoites, while GlcT-1 (ceramide-glucosyl transferase) transcript is expressed predominantly in encysting cells. As a result of SPT-2 gene upregulation in trophozoites, we hypothesize that 3-keto-sphinganine, which is synthesized by SPT and required for actin polymerization and endocytosis, increases the endocytic traffic in trophozoites. For cyst wall biosynthesis, however, we hypothesize that exogenous ceramide is taken up by Giardia and used as a scaffold to assemble complex saccharide-containing ceramide/sphingolipids with the help of the GlcT-1 enzyme (encoded by the GlcT-1 gene). To test these hypotheses we propose the following aims, i.e., Aim-1: Determine whether SPT-2 and GlcT-1 genes and enzymes that are differentially regulated in excysting and encysting Giardia; Aim-2: Determine whether post-transcriptional silencing of SPT-2 and GlcT-1 genes will interrupt the excystation-encystation cycle; and Aim-3: Determine whether SPT-2 and GlcT-1 mRNA or gene products are essential for ceramide endocytosis and metabolism in trophozoites, and synthesis of encystation-specific vesicles and cyst wall in the encysting cell. These studies will yield valuable information regarding the role of sphingolipids in regulating the giardial life cycle, and will lay the foundation for future efforts to develop new therapies against this waterborne pathogen.