Oligosaccharides to Prevent Infectious Diarrhea

Glycosyn, Llc, Medford MA

Investigators

Abstract

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].