A mutagenesis study of the antibiotic plectasin and the implications on its mode of action against MRSA

Abstract

The threat of antimicrobial resistance (AMR) is becoming a greater threat to the world’s healthcare. To combat AMR, there is a need to search and develop novel antibiotics. Plectasin is such novel antibiotic. It targets lipid II (LII), which is often referred to as the ‘Achilles heel’ of the cell. LII is an indispensable for the bacterium’s survival. It is the major building block for the bacterial peptidoglycan network and once disturbed, leads to rapid cell death. Plectasin is a cysteine-stabilized αβ defensin and a derivative of plectasin, NZ2114, is a triple mutant that has dramatically increased activity against methicillin-resistance Staphylococcus aureus, one of the most dangerous drug-pathogen combinations. The three mutations (D9N, M13L, Q14R) in NZ2114 however do not overlap with the suggested LII binding site, and no rationale why these mutations increase the efficacy against MRSA could be found thus far. Here, we investigated the effect of the three mutations on the efficacy of plectasin against MRSA, MSSA and Staphylococcus simulans using a combination of solution NMR, bacterial killing assays, and isothermal titration calorimetry (ITC) experiments. We found that mutations D9N and Q14R drastically increase plectasin’s activity against MRSA. The D9N mutation causes many chemical shift perturbations (CSP), specifically in the anionic patch and β-loop region that bind Ca2+ and W8 and F35 which are suggested to bind the sugars of LII. Mutations M13L and Q14R do not cause CSP in the Ca2+ binding site, but do cause CSP in the α-helix, which is responsible for oligomerization of plectasin.