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: 8291202
• 负责人: Siddhartha Das
• 金额: $ 42.43万
• 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/8291202
• 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是肠道感染的原因。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性包囊传播。包囊在通过人胃的过程中暴露于胃酸会触发脱囊，而滋养体定植的小肠中的因素会诱导包囊形成或包囊形成。包囊形成的标志是包囊特异性囊泡（ESV）的生物发生，ESV运输囊壁物质，随后与质膜融合并形成囊壁。然而，目前尚不清楚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。从我们的实验室的结果表明，贾第虫表达较少的鞘脂生物合成基因，这是在encystation差异调节。葡萄糖神经酰胺转移酶-1（glucosylceramide transferase-1，gGlcT 1）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，gGlcT 1的敲低干扰ESV形成，导致产生存活力降低的隐蔽性包囊。基于这些观察结果，我们假设gGlcT 1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是必需的。在特定目标1中，将使用gGlcT 1过表达和敲低贾第虫研究gGlcT 1调节脂质稳态和ESV生物发生的机制。我们将确定gGlcT 1是否与鞘脂生物合成途径的其他酶协调来调节ESV的形成。将通过质谱法和分子/细胞方法鉴定参与功能性ESV组装的脂质组分。在特定目标2中，将确定gGlcT 1在调节囊肿形态、活力和感染性方面的作用。将分别在体外脱囊和体内动物模型中测试通过gGlcT 1敲减产生的隐蔽囊肿的生物活性和感染性。将确定改变囊肿形态和活力的囊壁成分。这项拟议的研究将确定gGlcT 1是贾第虫形成囊肿的一种新调节剂，并可用于开发新的治疗方法来控制贾第虫病，每年影响全球数百万儿童。