Oligosaccharides to Prevent Infectious Diarrhea

描述(由申请人提供)：病原体与宿主细胞的黏附是感染的第一步，通常由特定的分子相互作用介导[1][2]。霍乱弧菌、致病性大肠杆菌(EPEC)、肠出血性大肠杆菌(EHEC)和诺沃克病毒的致病菌株是人类感染性腹泻的主要细菌和病毒原因[3]，它们通过与1(1，2)岩藻糖化细胞受体[4][5]结合，附着在肠道上皮细胞表面。1(1，2)岩藻糖化多糖在体外和体内都被证明能有效地防止这些病原体的结合和感染，它在人类母乳中含量丰富[6][7]。因此，这些分子代表了一种潜在的预防感染性腹泻的新类别的试剂，这种疾病每年导致全球200多万人死亡[8]。然而，生产足够数量的1(1，2)岩藻糖化多糖作为抗感染剂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]。体外酶法合成也是可能的，但受限于对昂贵的核苷酸-糖前体的要求。S的宏伟目标是通过微生物发酵低成本地批量生产1(1，2)岩藻糖化多糖，并设想了三类潜在的抗感染产品：1)纯化的1(1，2)岩藻糖化低聚糖，2)在细胞表面表达1(1，2)岩藻糖基化的酵母菌株，以及3)纯化的1(1，2)岩藻糖化糖蛋白。[本申请中概述的研究目标是在乳酸克鲁维酵母中生产第一个产品类别的示例，即纯化的1(1，2)岩藻糖基低聚糖，22-岩藻糖基乳糖(22-FL)，足够的量来测试该分子&lt；S作为单一制剂在体外和体内感染模型中的疗效。乳酸克鲁维酵母中的多糖合成途径将通过内源基因操作和编码所需活性的异源基因的引入相结合而被改造。具体地说，K.lactis将被改造成合成关键的前体糖GDP-岩藻糖，随后在细胞质中制造22-岩藻糖基乳糖。随后的目标是提高从乳酸钾中回收的2-岩藻糖基乳糖的产量，使其接近商业化所需的水平。为了实现这一目标，将通过控制合成酶和前体池的细胞水平来增加2；-岩藻糖基乳糖的产量，在生物反应器条件下平衡2；-FL产量与整体细胞活力和生长性能。] 公共卫生相关性：在世界范围内，感染性腹泻约占5岁以下儿童全部死亡的20%，每年估计有200万人死亡[8]。在发展中国家，细菌感染导致的腹泻病例占所有病例的50%以上，其中由弯曲杆菌和致泻性大肠杆菌引起的感染约占一半。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，每年导致4亿至5亿例腹泻。到目前为止，弯曲杆菌感染的发病率最高的是5岁的儿童[13][14][15]。不幸的是，细菌性腹泻的预防和治疗选择有限。疫苗目前无法获得，如果开发出来，将是昂贵的，而且在未得到满足需求最高的农村贫困人口中可获得的数量有限。此外，疫苗通常是病原体特有的，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有问题，因为这种使用正在推动耐药菌株的出现。例如，临床分离的弯曲杆菌现在经常对喹诺酮类药物产生抗药性[16]，红霉素抗药性菌株正在迅速出现[17]。传统的抗菌剂旨在抑制病原体&S的复制和生长，但它们并不会使病原体&S的环境生态位变得不可用。因此，新出现的耐药菌株很容易增殖和传播。需要研究开发既具有广泛作用又使用不同方法来避免耐药性发展的新型抗感染剂；例如，本申请中描述的新型抗粘附剂。这里描述的抗粘附性1(1，2)岩藻糖化低聚糖不会导致耐药性，因为它们不会通过仅仅剥夺病原体的环境生态位来对病原体施加选择性压力。此外，这些抗感染药物将同时针对多种肠道病原体，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].