GIARDIA ENCYSTMENT: CONTROL OF GA1NAC SYNTHESIS

贾第鞭毛虫病在美国和世界上许多其他国家都是一种主要的肠道疾病。其生命周期简单而直接，需要一个附着在宿主肠道微绒毛刷状边缘的营养滋养体和一个通过粪-口途径在宿主之间传播的囊肿。在胆汁诱导的囊肿形成（包囊）过程中，贾第鞭毛虫滋养体形成了富含囊壁特异性糖n -乙酰半乳糖胺（GalNAC，一种新型的[GalNAC β 1产3 GalNAC]n均聚物）的保护性囊壁细丝。GalNAc在非囊化的营养体中未检测到，在囊化过程中由葡萄糖通过五种可诱导的不可沉淀酶的活性合成：葡萄糖胺6-磷酸异构酶（GPI）、葡萄糖胺6-磷酸n-乙酰化酶（GlcNPA）、磷酸乙酰氨基葡萄糖变化酶（PAG1cNM）、UDP-N-乙酰氨基葡萄糖焦磷酸化酶（UDP-GlcNAcPP）和UDP-N-乙酰氨基葡萄糖4'外聚酶（UDP-GlcNAcE）。我们实验室的目标是深入研究酶的调控，分子生物学的控制，以及可能开发的新的化疗药物，可以针对这一途径，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已被纯化（或部分纯化）并鉴定，GPI和UDP-GlcNAcPP已被克隆并测序。我们还表明GPI受转录调控，而UDP-GlcNAcPP是组成性的，但单向激活GPI的合成代谢产物葡萄糖胺6-PO4合成GalNAc。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被纯化或表征的酶。因此，我们计划使用结合酶分析的分子技术来确定1)GlcNPA、PAGlcNM和UDP-GlcNAcE活性是在转录水平（如GPI的情况）还是在转录后水平（如贾第鞭虫的UDP-N-乙酰氨基葡萄糖焦磷酸化酶的情况）诱导，2)确定GlcNPA、PAG1cNM、和UDP-GlcNAcE是GalNAc合成途径中的调节酶，通过纯化（或表达）这些酶并表征它们的酶动力学，以及可能的激活剂和抑制剂。

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.