GIARDIA ENCYSTMENT: CONTROL OF GA1NAC SYNTHESIS

贾第鞭毛虫包囊：GA1NAC 合成的控制

• 批准号: 6157624
• 负责人: EDWARD L JARROLL
• 金额: $ 15.29万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-15 至 2004-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6157624
• 关键词: Giardia; N acetylgalactosamine; bacteria characteristic; bacteria infection mechanism; carbohydrate biosynthesis; enzyme activity; genetic regulation; genetic transcription; protein purification; protein sequence

Giardia causes is a major intestinal illness in the United States as well as many other countries worldwide. The life cycle is simple and direct requiring a vegetative trophozoite which attaches to the microvillus brush border of the host's intestine and a cyst which passes from host to host by the fecal-oral route. During cyst formation (encystment) induced by bile, Giardia trophozoites form a protective cyst wall filament rich in the cyst wall specific sugar N-acetylgalactosamine (GalNAC, as a novel [GalNAC beta1 yields 3 GalNAc]n homopolymer). GalNAc, undetected in non-encysting trophozoites, is synthesized from glucose during encystment by the activity of five inducible, nonsedimentable enzymes: Glucosamine 6-phosphate isomerase, GPI, glucosamine 6-phosphate N-acetylase (GlcNPA), phosphoacetylglucosamine mutase (PAG1cNM), UDP-N- acetylglucosamine pyrophosphorylase (UDP-GlcNAcPP), and UDP-N- acetylglucosamine 4' epimerase (UDP-GlcNAcE). The goal of our laboratories is an in-depth study of enzyme regulation for, the molecular biology for the control of, and the possible development of new chemotherapeutic agents that can target this pathway thus acting to prevent the formation of cysts and thus aiding in the control of giardiasis. To date GPI, UDP-GlcNAcPP and CWS have been purified (or partially purified) and characterized, and GPI and UDP-GlcNAcPP have been cloned and sequenced. We have shown also that GPI is transcriptionally regulated while UDP-GlcNAcPP is constitutive but unidirectionally activated toward GalNAc synthesis by glucosamine 6-PO4 the anabolic product of GPI. Before more in depth studies of the regulation of this pathway can be undertaken, it is essential to understand more about the three enzymes which have not yet been purified or characterized. Thus, we plan to use molecular techniques coupled with enzyme analyses to 1) determine whether GlcNPA, PAGlcNM, and UDP-GlcNAcE activities are induced at a transcriptional level (as is the case with GPI) or at a post- transcriptional level (as is the case with Giardia's UDP-N- acetylglucosamine pyrophosphorylase), and 2) determine if GlcNPA, PAG1cNM, and UDP-GlcNAcE are regulatory enzymes in the GalNAc synthetic pathway by purifying (or expressing) these enzymes and characterizing them with respect to enzyme kinetics, and possible activators and inhibitors.

贾第虫病是美国和世界上许多其他国家的一种主要肠道疾病。 其生活史简单而直接，需要一个营养体附着在宿主肠道的微绒毛刷状缘上，并需要一个包囊通过粪口途径在宿主之间传播。 在胆汁诱导的包囊形成（包囊形成）期间，贾第虫滋养体形成富含囊壁特异性糖N-乙酰半乳糖胺（GalNAC，作为一种新型[GalNAC β 1产生3 GalNAc]n均聚物）的保护性囊壁细丝。 GalNAc在非成囊滋养体中未检测到，在成囊期间通过5种可诱导的、不可沉淀的酶的活性由葡萄糖合成：葡萄糖胺6-磷酸异构酶、GPI、葡萄糖胺6-磷酸N-乙酰化酶（GlcNPA）、磷酸乙酰葡萄糖胺N-乙酰化酶（PAG 1cNM）、UDP-N-乙酰葡萄糖胺焦磷酸化酶（UDP-GlcNAcPP）和UDP-N-乙酰葡萄糖胺4'差向异构酶（UDP-GlcNAcE）。 我们实验室的目标是深入研究酶的调节，控制的分子生物学，以及可能开发新的化疗药物，这些药物可以靶向这一途径，从而防止囊肿的形成，从而帮助控制贾第虫病。 迄今为止，GPI、UDP-GlcNAcPP和CWS已经被纯化（或部分纯化）和表征，并且GPI和UDP-GlcNAcPP已经被克隆和测序。 我们还表明，GPI是转录调节，而UDP-GlcNAcPP是组成型的，但通过GPI的合成代谢产物葡糖胺6-PO 4单向激活GalNAc合成。 在对这一途径的调控进行更深入的研究之前，有必要更多地了解这三种尚未纯化或表征的酶。 因此，我们计划使用分子技术结合酶分析来1）确定GlcNPA、PAGlcNM和UDP-GlcNAcE活性是否在转录水平上被诱导（与GPI的情况一样）或在转录后水平（如贾第虫的UDP-N-乙酰葡糖胺焦磷酸化酶的情况），和2）确定GlcNPA，PAG 1cNM，和UDP-GlcNAcE是GalNAc合成途径中的调节酶，通过纯化（或表达）这些酶并在酶动力学方面表征它们，以及可能的激活剂和抑制剂。