NOVEL NEW ENZYME AND PRODUCT FROM ENCYSTING GIARDIA

贾第鞭毛虫（Giardia pestilalis）（syn. G. lamblia，G.是一种致病性的 原生动物寄生虫，在美国和国外，是一种主要的 水源性疾病暴发、托儿所或日托中肠道疾病的原因 设施和同性恋者疾病的粪口传播 当活的包囊被摄入时发生， 小肠上部出现两个滋养体。 滋养体定植在小肠中，至少部分在 在胆汁的刺激下，它们形成新的囊肿（包囊）， 粪便在包囊形成过程中，贾第虫滋养体合成一种 丝状孢囊壁，大部分由氨基组成 糖，N-乙酰半乳糖胺（GalNAc），在 非包囊滋养体。 为了实现这一目标，贾第虫 滋养体使用诱导酶的合成途径来转化 内源性葡萄糖转化为GalNAc。这条道路不仅在他们的 从滋养体到包囊的细胞分化，但代表了一种潜在的 对贾第虫的化疗攻击点。几种药物 目前被用来治疗贾第虫病和许多有不愉快的一面 方面的影响. 一些药物，包括最常用的药物甲硝唑， 有被淘汰的危险 虽然对甲硝唑的耐药性 在实践中尚未成为问题，体内和体外抗性已经 这是一个潜在的问题，因此存在一个潜在的问题。 因为我们最近才发现诱导型GalNAc合成物 在贾第鞭毛虫的途径，很少有人知道的调控或遗传控制 事实上，自从发现了这个途径， 我们还发现了一种以前未描述的酶活性， 称为“囊壁合成酶”。 “囊壁合成酶”活性 似乎将GalNAc固定在组成外层的不溶性细丝中， 贾第虫囊壁。 我们在这一建议中的意图是继续 研究这一途径，1）纯化，表征和定位 可诱导的颗粒状“囊壁合成酶”，和2）确定 其未表征产品的化学性质，可能是 贾第虫囊壁的主要成分。 我们将使用酶， 蛋白纯化技术，以达到酶的目的和标准 碳水化合物的分析方法，以实现化学分析的 酶的产物。由于缺乏关于 在大多数原生动物包囊机制，详细信息 这里产生的将提供关于一个关键代谢的重要信息， 途径至关重要贾第虫包囊和传播（也许有关 其他产孢原生动物），并可能为我们提供 这些信息对改善未来的化疗很重要。

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is a pathogenic protozoan parasite, and in the United States and abroad, is a leading cause of intestinal disease in waterborne outbreaks, nursery or day-care facilities, and homosexuals. Fecal-oral transmission of the disease occurs when viable cysts are ingested, and excystation occurs in the upper small intestine with the emergence of two trophozoites. Trophozoites colonize the small intestine where, at least in part stimulated by bile, they form new cysts (encystment) which are shed in the feces. During encystment, Giardia trophozoites synthesize a filamentous cyst wall which is composed, in large part, of the amino sugar, N-acetylgalactosamine (GalNAc) which is undetectable in non-encysting trophozoites. In order to accomplish this, Giardia trophozoites use a synthetic pathway of inducible enzymes to convert endogenous glucose to GalNAc. This pathway is not only paramount in their cytodifferentiation from trophozoite to cyst, but represents a potential point of chemotherapeutic attack against Giardia. Several drugs are currently being used to treat giardiasis and many have unpleasant side effects. Some, including the most frequently used drug, metronidazole, are at risk of becoming obsolete. While resistance to metronidazole has not yet become a problem in practice, in vivo and in vitro resistance has been demonstrated and thus a potential problem exists. Since we have only recently discovered the inducible GalNAc synthetic pathway in Giardia, little is known of the regulation or genetic control of the enzymes that comprise it. Indeed, since discovery of this pathway, we have also found a previously undescribed enzyme activity, tentatively referred to as "cyst wall synthetase". "Cyst wall synthetase" activity appears to fix GalNAc into the insoluble filaments that compose the outer cyst wall of Giardia. It is our intent in this proposal to continue studying this pathway by 1) purifying, characterizing and localizing the inducible, particulate "cyst wall synthetase" and 2) determining the chemical nature of its uncharacterized product which is likely to be a major component of the Giardia cyst wall. We will employ enzyme and protein purification techniques to achieve the enzymatic aim and standard methods of carbohydrate analyses to achieve the chemical analysis of the enzyme's product. Since there is a paucity of information of the mechanisms of encystment in most protozoa, the detailed information generated here will provide important information about a key metabolic pathway vital to Giardia encystment and transmission (and perhaps related to that in other cyst~producing protozoa), and will likely provide us with information important to improving chemotherapy in the future.