Engineered Probiotic Yeast to Prevent Infectious Diarrhea

描述（由申请人提供）：工程益生菌酵母预防感染性腹泻。病原体附着在宿主细胞上是感染必不可少的第一步，通常由特定的分子相互作用介导[1,2]。致病性弯曲杆菌和诺瓦克病毒是导致人类感染性腹泻的主要细菌和病毒，它们通过与？（1,2）聚焦细胞受体[4,5]。?？（1,2）浓缩聚糖在人母乳中含量丰富[6,7]，在体内和体外均已被证明能有效地阻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传染性腹泻潜力的新制剂，这种疾病每年在全球造成200多万人死亡。然而，生产？？（1,2）聚焦聚糖作为抗感染药物的足够数量来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、产品杂质和高总体成本的限制[9-11]。体外酶合成也是可能的，但由于需要昂贵的核苷酸-糖前体而受到限制。格瑞辛公司的主要目标是开发制造？（1,2）通过微生物发酵廉价和批量浓缩的聚糖，以及三类潜在的抗感染产品：1)纯化的？？（1,2）聚焦寡糖；(2)表达？（1,2）在细胞表面聚焦多糖，(3)纯化？（1,2）聚焦糖蛋白。本申请中概述的研究目标是产生并测试作为益生菌的第二类产品的例子，即表达？？（1,2）在其细胞表面聚焦多糖。通过内源基因操作和引入编码所需活性的外源基因，对常见乳酵母克鲁维酵母的聚糖合成途径进行工程化。具体来说，K.lactis将被设计合成关键的前体糖，GDP-焦糖，将GDP-焦糖和udp -半乳糖运输到高尔基体的能力，以及精心制作的能力？？（1,2）聚焦在细胞表面的聚糖。表面结合？？将优化（1,2）聚焦聚糖。工程酵母将在体外进行病原体结合评估，并在体内弯曲杆菌感染动物模型中作为益生菌使用。公共卫生相关性：在世界范围内，感染性腹泻约占5岁以下儿童死亡总数的20%，估计每年造成250万人死亡。在美国，感染性腹泻的年发病率超过2亿例，每年约有90万人住院，5000人死亡[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，最大单一细菌原因是弯曲杆菌感染。表面？？（1,2）聚焦型益生菌乳酸菌（K.lactis）是本次应用的主题，将针对诺瓦克样病毒和空肠梭菌，以及其他聚焦结合型肠道病原体。

DESCRIPTION (provided by applicant): Engineered Probiotic Yeast to Prevent Infectious Diarrhea. 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 ?(1,2) fucosylated cellular receptors [4,5]. ??(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 ??(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 ?(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified ??(1,2) fucosylated oligosaccharides, 2) yeast strains expressing ?(1,2) fucosylated glycans on their cell surface, and 3) purified ?(1,2) fucosylated glycoproteins. The goal of the studies outlined in this application is to generate and test as a probiotic an example of the second of these product classes, namely an engineered yeast strain expressing ??(1,2) fucosylated glycans on its cell surface. 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, the ability to transport both GDP-fucose and UDP-galactose to the Golgi, and the ability to elaborate ??(1,2) fucosylated glycans on the cell surface. Yields of surface-bound ??(1,2) fucosylated glycans will be optimized. The engineered yeast will be evaluated for pathogen binding in vitro and for use as a probiotic in an in vivo animal model of Campylobacter 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 ??(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.