Inhibition and Structural Insights into the Mechanism of Peptidoglycan O-Acetylation in Neisseria gonorrhoeae

The peptidoglycan (PG) sacculus, which is unique to bacterial cell walls, fulfills a crucial role in maintaining cellular viability. It is no coincidence that PG, and the associated cellular machinery, represents a common target for antibacterial agents as well as the host immune systems as a decline in integrity of this cellular armour is often fatal. Bacteria however have evolved to minimize the risk of this “Achilles heel” through the chemical modification of PG. O-Acetylation of the C-6-hydroxyl of N-acetylmuramic acid (MurNAc) is commonly found within many human pathogens including Neisseria gonorrhoeae, Campylobacter, and Staphylococcus aureus; all of which were identified by the World Health Organisation as either “Urgent” or “Serious Threats” in a 2019 report on antibiotic resistance. O-Acetylation increases pathogenicity due to its ability to inhibit the activity of lysozyme, a first line of defense used by mammalian immune systems. In Gram-negative bacteria it holds additional importance as a means to control endogenous autolysins. Inhibition of this modification presents an attractive target for the development of a new class of antimicrobials which would exploit a unique antivirulence strategy in rendering the bacteria susceptible to not only the host immune system but also autolytic activity. In Gram-negative bacteria, O-acetylation is achieved through the use of a two-component system of O-acetyltransferases (Pats). PatA, a transmembrane protein, shuttles an acetyl group across the cytoplasmic membrane into the periplasm where PatB, the dedicated PG O-acetyltransferase, directly modifies the C-6-hydroxyl of MurNAc within the existing sacculus. The purpose of this study was to validate PatB from N. gonorrhoeae as a novel antivirulence target which was accomplished via the identification and characterization of the first inhibitors of a Gram-negative PG O-acetyltransferase. To help guide the development and characterization of future inhibitors, the structure of PatB was solved using X-ray crystallographic techniques. Within the structure, a unique and unpredicted feature was identified which led to additional investigation into the association between PatB and its native substrate, the PG sacculus. The structure of PatA from N. gonorrhoeae was also investigated in silico and the first complete 3D model and proposed molecular mechanism are presented.