Bacterial cell wall modification by glycolipid probes

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. The structures of mammalian glycans can be perturbed through metabolic incorporation, in which non‐natural sugars are taken up by cells, converted to nucleotide‐sugar intermediates, and incorporated via biosynthetic pathways. Applications of metabolic incorporation in bacteria are complicated by the diversity of pathways bacteria use to acquire, metabolize, and catabolize monosaccharide intermediates. We developed a strategy to alter glycan structure that avoids intracellular processing using lipid‐linked glycans, intermediates directly used in glycan biosynthesis. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis , organisms that serve as models of Mycobacterium tuberculosis . Using a C. glutamicum mutant that lacks arabinan, we identified glycolipid donors whose addition restores cell wall arabinan, demonstrating that non‐natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. Our lipid‐based probe could rescue wild type cells treated with an inhibitor of cell wall biosynthesis. We then focused on incorporating derivatives with chemical handles for labeling such that bacterial glycans can be captured and visualized. To this end, we have generated the first probe to label arabinose‐containing structures in mycobacteria and corynebacteria. Support or Funding Information NIH: RO1‐AI063596, NSERC PGS‐D