Sphingolipids as Potential Targets for Anti-Giardial Therapy

鞘脂作为抗贾第虫疗法的潜在靶点

• 批准号: 7282236
• 负责人: Siddhartha Das
• 金额: $ 14.13万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282236
• 关键词: Actins; Affect; Anabolism; Apoptosis; Biochemical; Biogenesis; Biological Assay; Cells; Ceramide glucosyltransferase; Ceramides; Clathrin; Complex; Confocal Microscopy; Cyst; Cytoskeleton; Disease; Double-Stranded RNA; Endocytosis; Endoplasmic Reticulum; Energy-Generating Resources; Enteral; Enzyme Gene; Enzymes; Eukaryotic Cell; Event; Foundations; Future; Gastric Acid; Gene Targeting; Genes; Giardia; Giardia lamblia; Glucosylceramides; Goals; High Pressure Liquid Chromatography; Human; Immunoblotting; Immunoprecipitation; Invasive; Life Cycle Stages; Mass Spectrum Analysis; Mastigophora; Measures; Membrane; Messenger RNA; Metabolic; Metabolic Pathway; Metabolism; Molecular; Molecular Analysis; Operative Surgical Procedures; Parasites; Pathway interactions; Personal Satisfaction; Plasmids; Play; Process; Production; Proteins; Resistance; Role; Serine; Signal Transduction; Small Intestines; Sphingolipids; Stomach; Testing; Transcript; Transfection; Transferase; Up-Regulation; Vesicle; Water; Yeasts; base; excystation; hammerhead ribozyme; membrane synthesis; novel; pathogen; polymerization; scaffold; sphinganine; trafficking; waterborne; waterborne infection

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.

贾第鞭毛虫是一种非侵入性寄生虫，可引起人类水源感染。这 有鞭毛的原生动物以两种形式存在，即滋养体和包囊。形态学 从包囊到滋养体的转化（脱囊）发生在胃中，而因素 小肠触发滋养体向囊肿（包囊）的转变。此次活动的主要目标 该提案旨在阐明鞘脂在调节包囊-脱囊循环中的新作用 贾第鞭毛虫。众所周知，鞘脂在信号传导、分化和细胞凋亡中发挥着重要作用 在所有真核细胞中。然而，贾第鞭毛虫合成鞘脂的能力有限，依赖于 用于能量生产和膜生物合成的外源性鞘脂供应。我们的研究 表明贾第鞭毛虫使用肌动蛋白/网格蛋白依赖性途径来导入和靶向神经酰胺，神经酰胺是 鞘脂的前体，形成内质网/核周膜。分子分析表明只有两种 鞘脂代谢基因在滋养体和包囊贾第鞭毛虫中差异表达。 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-葡萄糖基转移酶）转录物主要在包囊细胞中表达。后果 SPT-2 基因在滋养体中上调，我们假设合成 3-酮-二氢鞘氨醇 通过 SPT 并需要肌动蛋白聚合和内吞作用，增加内吞流量 滋养体。然而，对于囊壁生物合成，我们假设外源神经酰胺被吸收 由贾第鞭毛虫制成，用作组装复杂的含糖神经酰胺/鞘脂的支架 在 GlcT-1 酶（由 GlcT-1 基因编码）的帮助下。为了检验这些假设，我们提出 以下目标，即 Aim-1：确定 SPT-2 和 GlcT-1 基因和酶是否 贾第鞭毛虫的去囊和包囊受到不同的调节； Aim-2：确定转录后是否 SPT-2 和 GlcT-1 基因的沉默将中断脱壳-包囊周期；和目标 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 滋养体中的代谢和代谢，以及包囊特异性囊泡和囊壁的合成 包囊细胞。 这些研究将产生有关鞘脂在调节神经系统中的作用的有价值的信息。 贾第虫生命周期，并将为未来开发针对该疾病的新疗法奠定基础 水传播的病原体。