Reversible Degradation of Peptidoglycan by Lytic Transglycosylases

Antimicrobial resistance continues to be a burden on the global healthcare system with an estimated cost of billions of dollars and millions of deaths each year. Recently, there has been little to no development on new antibiotics to treat bacterial infection, and any that are developed become resisted to within a few years. Both Gram-positive and Gram-negative bacteria contain a mesh-like layer called peptidoglycan (PG) that surrounds their cells providing strength, cell shape, and protection from their environments. This layer is a polymer of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) connected via a b-1,4-glycosidic bond, with each strand being cross-linked to another through a peptide stem attached to MurNAc. Lytic transglycosylases (LTs) are bacterial enzymes that cleave the glycosidic bond producing GlcNAc and a unique 1,6-anhydroMurNAc product, unlike other muramidases such as lysozyme, which produce GlcNAc and MurNAc. LTs are known to cleave PG in order to make space for extracellular components, and to aid in cellular growth, but it is not clear why they specifically generate the unique 1,6-anhydroMurNAc reaction product. The research presented in this thesis explores the hypothesis that LTs also catalyze a reverse reaction, re-ligating PG together in order to maintain its integrity. Neisseria meningitidis LtgA was used as the model enzyme because it is conveniently active on chitooligosaccharides which are commercially available. Two main assays were developed to test the hypothesis. The first utilized a fluorescent dye to label PG, and thereby follow the enzymatic production of reaction products. The second assay used chitopentaose as a soluble substrate in an HPLC-based assay to monitor the production of products with higher degrees of polymerization. Both assays demonstrated the use of 1,6- anhydro amino sugars by LtgA for transglycosylation reactions. This provided a proof-of-concept for this hypothesis and that the assays could serve to further characterize the reaction and potentially be used to develop the LTs as a new antibiotic target.