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. Virulent Campylobacter species and pathogenic strains of Norwalk virus, the leading bacterial and viral causes of human infectious diarrhea, adhere to gut epithelial surfaces through binding to fucosylated cellular receptors. (1,2) fucosylated glycans, which are abundant in human breast milk, have been shown both in vitro and in vivo effectively to prevent binding and infection by these pathogens. 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. 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. In vitro enzymatic syntheses are also possible but are limited by a requirement for expensive nucleotide-sugar precursors. <P> 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. <P> 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. <P> PUBLIC HEALTH RELEVANCE: Worldwide, infectious diarrhea is responsible for approximately 20% of all mortality in children under the age of 5, and for an estimated 2.5 millions deaths annually. 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. 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.