Sphingolipid and Mechanism of Cyst Formation by Giardia

鞘脂和贾第鞭毛虫囊肿形成机制

• 批准号: 8680128
• 负责人: Siddhartha Das
• 金额: $ 41.96万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8680128
• 关键词: 1-Propanol; Abbreviations; Address; Adverse effects; Affect; Anabolism; Animal Model; Biochemical; Biogenesis; Biological; Cell membrane; Cells; Ceramide glucosyltransferase; Child; Cyst; Developed Countries; Developing Countries; Development; Diarrhea; Disease; Drug Compounding; Drug resistance; Enzymes; Eukaryota; Fatty Acids; Feces; Food; Gastric Acid; Generations; Genes; Giardia; Giardia lamblia; Giardiasis; Glucosylceramides; Glycosphingolipids; Goals; Homeostasis; Human; In Vitro; Infection; Intestines; Label; Laboratories; Lipids; Malabsorption Syndromes; Mass Spectrum Analysis; Metabolic Pathway; Methodology; Metronidazole; Molecular; Morphology; Oligonucleotides; Parasites; Pathogenesis; Pathway interactions; Phospholipids; Process; Production; Proteins; Resistance; Role; Small Intestines; Sphingolipids; Sphingomyelins; Stomach; Testing; Transferase; Transport Vesicles; Vesicle; Water; acid sphingomyelinase; analog; base; biodefense; design; disorder control; effective therapy; excystation; in vivo; novel; overexpression; research study; serine palmitoyltransferase; transmission process; uptake; waterborne

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.

描述（由申请人提供）：蓝氏贾第鞭毛虫是一种水生寄生虫，在发达国家和发展中国家都会引起肠道感染。贾第鞭毛虫病也是一种人畜共患疾病，通过受污染的水通过感染性包囊传播。包囊在通过人胃的过程中暴露于胃酸会引发包囊作用，而滋养体定植的小肠中的因素会诱导包囊或包囊形成。包囊的标志是包囊特异性囊泡（ESV）的生物发生，它运输囊壁物质，随后与质膜融合并放下囊壁。然而，尚不清楚 ESV 生物发生是如何调节的以及如何产生有活力或感染性的包囊。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，这些基因在成囊过程中受到差异性调节。葡萄糖神经酰胺转移酶 1 (gGlcT1) 是 SL 生物合成的重要酶之一，其过度表达会产生扩大和聚集的 ESV，并改变细胞脂质稳态。另一方面，gGlcT1 的敲除会干扰 ESV 的形成，从而导致活力降低的隐性囊肿的产生。基于这些观察，我们假设 gGlcT1 的调节表达对于 ESV 生物发生和传播疾病的感染性包囊的产生至关重要。在具体目标 1 中，将使用 gGlcT1 过表达和敲低贾第鞭毛虫来研究 gGlcT1 调节脂质稳态和 ESV 生物发生的机制。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形成。功能性 ESV 组装中涉及的脂质成分将通过质谱和分子/细胞方法进行鉴定。在具体目标 2 中，将确定 gGlcT1 在调节囊肿形态、活力和感染性中的作用。 gGlcT1敲低产生的隐性包囊的生物活性和感染性将分别在体外排泄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肿壁成分。拟议的研究将确定 gGlcT1 是贾第鞭毛虫包囊形成的新型调节剂，可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。