Methods for the Study of Galactofuranose in Mycobacteria

Abstract

Despite the energy costs associated with deploying furanose sugars over their more stable pyranose counterparts, galactofuranose is prevalent across nature from commensals to human pathogens. However, it is conspicuously absent from human cells, thus establishing its biosynthetic pathways as important potential drug targets. Our knowledge about the biological roles of galactofuranose in cells is hampered by a dearth of methods by which to study it. Access to glycans containing galactofuranose is limited by the unfavorable equilibrium between pyranose and furanose forms of the sugar, leading to low-yielding and synthetically arduous routes to galactofuranose glycans and its high-energy nucleotide sugar donor, used in biochemical experiments to probe the activity and kinetics of galactofuranose glycosyltransferases. Moreover, study of carbohydrate structures within the cell is limited by the lack of methods to selectively modify glycans with functional handles. Finally, study of the biosynthetic machinery for galactofuranose biosynthesis and inhibitors thereof is limited by their relatively weak affinities for their ligands, providing a challenge for selective chemical probes. In this work, we describe three methods to address these challenges and expedite the study of galactofuranose-containing glycans, their biological function, and their biosynthetic machinery. First, we developed a method to produce the rare high-energy sugar donor UDP-galactofuranose in situ for facile preparation of the mycobacterial galactan utilizing the sugar mutase UDP-galactopyranose mutase. We used this method to generate up to 10 milligrams of polymer and demonstrated that it could be selectively functionalized. Second, we leveraged the rapidly expanding set of biosynthetic probes of the mycobacterial cell wall to rapidly characterize intracellular distances between distinct layers of the cell wall in Mycobacterium tuberculosis model organisms Corynebacterium glutamicum and Mycobacterium smegmatis using fluorescence resonance energy transfer. We evaluated strains with varying galactan structures and compared our data to previous characterization of the cell wall to assess the method’s utility. Finally, we characterized the kinetics of a mild electrophile, the squaric ester, and assessed its utility in selectively binding and modifying a key galactofuranosyl transferase involved in mycobacterial cell wall biosynthesis. Taken together, these findings present a suite of methods to expedite the exploration of galactofuranose structure and function within a relevant pathogen and lays groundwork for further study of galactofuranose across other organisms.