Oligosaccharides to Prevent Infectious Diarrhea

描述（由申请人提供）：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]。毒性弯曲杆菌属、霍乱弧菌、肠致病性大肠杆菌（EPEC）、肠出血性大肠杆菌（EHEC）和诺瓦克病毒的致病性菌株（人类感染性腹泻的主要细菌和病毒原因[3]）通过结合1（1，2）岩藻糖基化细胞受体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，生产足够数量的1（1，2）岩藻糖基化聚糖作为抗感染药物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，产品杂质和高总成本的限制[9][10][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.本发明的广泛目标是开发通过微生物发酵廉价且大量生产1（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1（1，2）岩藻糖基化低聚糖，2）在其细胞表面上表达1（1，2）岩藻糖基化聚糖的酵母菌株，和3）纯化的1（1，2）岩藻糖基化糖蛋白。本申请中概述的研究的目标是在乳酵母乳酸克鲁维酵母中产生这些产品类别中的第一类的实例，即纯化的1（1，2）岩藻糖基化低聚糖，22-岩藻糖基乳糖（22-FL），其量足以测试该分子作为体外和体内感染模型中的单一药剂的功效。乳酸克鲁维酵母中的聚糖合成途径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。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，随后在细胞质中产生22-岩藻糖基乳糖。随后的目标是增加从乳酸克雷伯氏菌回收的2 α-岩藻糖基乳糖的产率，以接近商业化所需的水平。为了实现这一点，将通过操纵合成酶和前体库的细胞水平来增加2;-岩藻糖基乳糖的生产，平衡2;-FL生产与生物反应器条件下的总体细胞活力和生长性能。] 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200万人死亡[8]。在发展中国家，细菌感染占所有腹泻病例的50%以上，其中弯曲杆菌和大肠杆菌感染约占一半。弯曲杆菌是发达国家和发展中国家经培养证实的细菌性胃肠炎的最常见原因，每年造成4亿至5亿例腹泻病例。到目前为止，弯曲杆菌感染的发病率最高的是5岁以下的儿童[13][14][15]。 不幸的是，细菌性腹泻的预防和治疗选择有限。目前没有疫苗，如果开发出来，将是昂贵的，而且在农村贫困人口中的供应有限，因为那里的需求得不到满足。此外，疫苗通常是病原体特异性的，但感染性腹泻可以由许多不同的病原体引起。使用抗生素治疗腹泻也变得越来越成问题，因为这种使用正在推动耐药菌株的出现。例如，弯曲杆菌的临床分离株现在通常对喹诺酮类药物具有耐药性[16]，而红霉素耐药菌株正在迅速出现[17]。传统的抗菌剂被设计成抑制病原体的复制和生长，但它们不能使病原体的环境生态位不可用。因此，新出现的耐药菌株很容易增殖和传播。需要研究开发新类别的抗感染剂，其既广泛作用又使用不同的方法来避免耐药性的发展;例如新型抗粘附剂，如本申请中描述的那些。本文所述的抗粘附1（1，2）岩藻糖基化低聚糖不会产生耐药性，因为它们仅通过剥夺病原体的环境生态位而对病原体不施加选择性压力。此外，这些抗感染药物将同时靶向多种肠道病原体，包括空肠弯曲菌[4]、ETEC大肠杆菌[18]、霍乱弧菌[19][4]和其他[5][20]。

DESCRIPTION (provided by applicant): Adherence of pathogens to their host cells is the obligatory first step of infection and is frequently mediated by specific molecular interactions [1][2]. Virulent Campylobacter species, Vibrio cholerae, enteropathogenic E.coli (EPEC), enterohemorrhagic E.coli (EHEC) 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 that is the cause annually of over 2 million deaths worldwide [8]. However the production of 1(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][10][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(1,2) fucosylated glycans cheaply and in bulk through microbial fermentation, and three classes of potential anti-infective products are envisaged: 1) purified 1(1,2) fucosylated oligosaccharides, 2) yeast strains expressing 1(1,2) fucosylated glycans on their cell surface, and 3) purified 1(1,2) fucosylated glycoproteins. [[[The goal of the studies outlined in this application are to produce in the dairy yeast Kluyveromyces lactis an example of the first of these product classes, namely a purified 1(1,2) fucosylated oligosaccharide, 22-fucosyllactose (22-FL), in sufficient amounts to test this molecule<s efficacy as a single agent in in vitro and in vivo infection models. Glycan synthetic pathways in 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 22-fucosyllactose in the cell cytoplasm. A subsequent goal is to increase the yield of 2<-fucolsyllactose recovered from K.lactis to approach levels that will be required for commercialization. To achieve this the production of 2<fucosyllactose will be increased by manipulating cellular levels of synthetic enzymes and precursor pools, balancing 2;-FL production with overall cell viability and growth performance under bioreactor conditions.]]] 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 million deaths annually[8]. In the developing world bacterial infections cause more than 50% of all cases of diarrhea, and of these, infections by Campylobacter and diarrheagenic E.coli together account for about half. Campylobacter is the most common cause of culture-proven bacterial gastroenteritis in both developed and developing countries, and is responsible for 400 to 500 million cases of diarrhea each year. By far the highest incidence of Campylobacter infections is in children <5 yrs of age[13][14][15]. Unfortunately, prevention and treatment options for bacterial diarrhea are limited. Vaccines are currently unavailable, and if developed, would be costly and of limited availability in rural poor populations where unmet need is highest. Moreover vaccines are typically pathogen-specific, but infectious diarrhea can be caused by numerous diverse pathogens. The use of antibiotics for treatment of diarrhea is also becoming increasingly problematic, since such use is driving the emergence of resistant strains. For example, clinical isolates of Campylobacter are now often resistant to quinolones [16] and erythromycin-resistant strains are rapidly emerging [17]. Conventional antimicrobial agents are designed to inhibit a pathogen<s replication and growth, yet they do nothing to make a pathogen<s environmental niche unavailable. Thus emerging resistant strains are readily able to proliferate and spread. Research is needed to develop new classes of anti-infective agent that are both broad-acting and that use a different approach to avoid the development of resistance; for example novel anti-adhesion agents such as those described in this application. The anti-adhesive 1(1,2) fucosylated oligosaccharides described here will not drive resistance, since they exert no selective pressure on pathogens by merely depriving them of their environmental niche. Moreover these anti-infectives will simultaneously target multiple enteropathogens, including C.jejuni [4], ETEC E.coli [18], Vibrio cholerae [19][4] and others [5][20].