Deciphering Helicobacter pylori's glycocode: uncovering and harnessing drug targets

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Antibiotic resistant bacterial pathogens cause more than 2.8 million new infections and 32,000 deaths each year in the US,1 indicating the ineffectiveness of our existing arsenal of antibiotics. Even when antibiotics are effective at eradicating infection, most are inherently nonspecific for pathogens and have unintended conse- quences on beneficial microbiota.2 Bacterial cell surface glycans are compelling drug targets as they contain exclusively bacterial structures that are critical for strain fitness and pathogenesis.6-8 Despite the importance of glycan biosynthesis as a virulence factor, the systematic study and inhibition of bacterial glycans remains chal- lenging due to their utilization of rare deoxy amino sugars and their production of products that are refractory to traditional glycan analysis. The deployment of chemical tools to study bacterial glycans is a crucial step toward developing new glycosylation-based strategies to eradicate pathogenic infections. My laboratory seeks to apply metabolic oligosaccharide engineering (MOE) tools to diverse microbial communities and to develop new tools to study and perturb glycan biosynthesis in the gastric pathogen Helicobacter pylori (Hp). The central hypothe- sis of the application is that MOE with unnatural monosaccharides, including rare bacterial monosaccharide analogs11 and fluorescent inhibitors, will facilitate profiling glycopatterns in complex microbial communities, tracking of bacterial glycan biosynthesis, and disarming of bacteria by perturbing their glycan armor. Our hy- pothesis has been formulated on the basis of strong preliminary data produced in my laboratory, including the application of MOE to install detectable reporters into bacterial glycans,9-11 thus permitting the discovery of a protein glycosylation system,12, 13 the identification of glycosylation genes,14 and the development of metabolic glycan inhibitors.15 The proposed work will reveal new targets of therapeutic intervention, resulting in innovative approaches to treat bacterial infection. The central hypothesis will be tested by pursuing three specific aims. In Aim 1, metabolic glycan reporters will be used to profile and perturb glycopatterns in complex microbial com- munities, revealing information about glycan structure, underlying glycosylation machinery, and the selectivity with which bacteria can be targeted based on their distinctive glycan coating. In Aim 2, fluorescent substrate- based monosaccharide analogs will be used to track glycan biosynthesis intermediates and end products in Hp glycosylation mutants versus wildtype strains, yielding insight into how Hp glycosylation enzymes act together to produce glycans. In Aim 3, metabolic glycan inhibitors will disrupt glycoprotein biosynthesis in Hp, allowing us to sensitize Hp to host immune attack and potentiate the use of existing antibiotics. The proposed work will uncover bacterial glycans from the human gut microbiome that are amenable to study and inhibition with un- natural sugars, yield a bacterial glycomics approach to study glycan construction, and probe the effect of al- tered glycan architecture on immune recognition and antibiotic efficacy. Broadly, this work will introduce ap- proaches to study and harness glycans of priority pathogens to create urgently needed antibiotics.