Oligosaccharides to Prevent Infectious Diarrhea

描述（由申请方提供）：病原体与其宿主细胞的粘附是感染的第一步，通常由特异性分子相互作用介导[1，2]。诺沃克病毒的毒性弯曲杆菌属物种和致病性菌株是人类感染性腹泻的主要细菌和病毒原因[3]，通过与1（1，2）岩藻糖基化细胞受体结合而粘附于肠道上皮表面[4，5]。1（1，2）岩藻糖基化聚糖在人类母乳中含量丰富[6，7]，已在体外和体内证明可有效防止这些病原体的结合和感染[4，5]。因此，这些分子代表了一类具有预防感染性腹泻潜力的新型药剂，感染性腹泻是全球每年超过200万人死亡的原因[8]。然而，作为抗感染剂的α（1，2）岩藻糖基化聚糖的生产量足以影响全球腹泻发病率仍然是一个重大挑战。化学合成是可能的，但受到立体特异性问题、产物杂质和高总成本的限制[9-11]。体外酶促合成也是可能的，但受限于对昂贵的核苷酸-糖前体的要求。Glycosyn Inc.的广泛目标是开发通过微生物发酵廉价和大量生产α（1，2）岩藻糖基化聚糖的方法，并且设想了三类潜在的抗感染产品：1）纯化的α（1，2）岩藻糖基化寡糖，2）在其细胞表面上表达α（1，2）岩藻糖基化聚糖的酵母菌株，和3）纯化的α（1，2）岩藻糖基化糖蛋白。本申请中概述的研究的目标是合成、纯化和测试作为营养补充剂的第一类产品的实例，即纯化的α（1，2）岩藻糖基化低聚糖，2-岩藻糖基乳糖（2FL）。将通过内源基因操作和引入编码所需活性的异源基因的组合来工程化普通乳酵母Kluyveromyces lactis中的聚糖合成途径。具体而言，乳酸克雷伯氏菌将被工程化以合成关键前体糖GDP-岩藻糖，并随后在细胞质中产生2-岩藻糖基乳糖。将优化产品收率，并制定提取和纯化方案。将在体外和体内感染模型中检测乳酸克雷伯菌中产生的纯化2-岩藻糖基乳糖的疗效。 公共卫生相关性：在世界范围内，感染性腹泻[12]占5岁以下儿童死亡率的约20%，估计每年有250万人死亡[8]。在美国，感染性腹泻的年发病率超过2亿例，每年造成约90万例住院和5,000例死亡[3，13]。诺瓦克样病毒感染是迄今为止美国感染性腹泻的最大单一原因，其中最大的单一细菌原因是弯曲杆菌属感染。作为本申请的主题的表面α（1，2）岩藻糖基化益生菌乳酸克雷伯氏菌将靶向诺瓦克样病毒和空肠弯曲杆菌，以及其它岩藻糖结合性肠道病原体。

DESCRIPTION (provided by applicant): Adherence of pathogens to their host cells is the obligatory first step in infection and is frequently mediated by specific molecular interactions [1, 2]. Virulent Campylobacter species and pathogenic strains of Norwalk virus, the leading bacterial and viral causes of human infectious diarrhea [3], adhere to gut epithelial surfaces through binding to 1(1, 2) fucosylated cellular receptors [4, 5]. 1(1, 2) fucosylated glycans, which are abundant in human breast milk [6, 7], have been shown both in vitro and in vivo effectively to prevent binding and infection by these pathogens [4,5]. These molecules therefore represent a new class of agent with potential to prevent infectious diarrhea, a condition which is the cause annually of over 2 million deaths worldwide [8]. However the production of a(1, 2) fucosylated glycans as anti-infective agents in sufficient quantities to impact global diarrhea incidence remains a significant challenge. Chemical syntheses are possible, but are limited by stereo-specificity issues, product impurities, and high overall cost [9-11]. In vitro enzymatic syntheses are also possible but are limited by a requirement for expensive nucleotide-sugar precursors. Glycosyn Inc.'s broad goal is to develop ways to manufacture a(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified a(1,2) fucosylated oligosaccharides, 2) yeast strains expressing a(1,2) fucosylated glycans on their cell surface, and 3) purified a(1,2) fucosylated glycoproteins. The goal of the studies outlined in this application are to synthesize, purify, and test as a nutritional supplement, an example of the first of these product classes, namely a purified a(1,2) fucosylated oligosaccharide, 2-fucosyllactose (2FL). Glycan synthetic pathways in the common dairy yeast Kluyveromyces lactis will be engineered through a combination of endogenous gene manipulation and the introduction of heterologous genes encoding desired activities. Specifically, K.lactis will be engineered to synthesize the key precursor sugar, GDP-fucose, and subsequently to make 2-fucosyllactose in the cell cytoplasm. Yields of product will be optimized, and extraction and purification schemes will be developed. Purified 2-fucosyllactose produced in K.lactis will be tested for efficacy both in vitro and in vivo in models of infection. PUBLIC HEALTH RELEVANCE: Worldwide, infectious diarrhea [12] is responsible for approximately 20% of all mortality in children under the age of 5, and for an estimated 2.5 millions deaths annually [8]. In the United States infectious diarrhea has an annual incidence of over 200 million cases and is responsible for approximately 900,000 hospitalizations and 5,000 deaths per year [3, 13]. Infection by Norwalk-like viruses is by far the single largest cause of infectious diarrhea in the US, with the single largest bacterial cause being infection by Campylobacter species. The surface a(1,2) fucosylated probiotic K.lactis that is the subject of this application will target both Norwalk-like viruses and C.jejuni, as well as other fucose-binding enteropathogens.