Nonactive-site mutations in S. aureus FabI that induce triclosan resistance

Abstract

The wide use of the antimicrobial agent/biocide, triclosan, promotes triclosan-resistant bacterial strains, including Staphylococcus aureus, as well as leads to accumulation in the aquatic and terrestrial environments. Knowledge of the molecular actions of triclosan on S. aureus is needed to understand the consequence of triclosan resistance and environmental accumulation of triclosan on S. aureus resistant strains, as well as to develop biphenyl ether analogs as antibiotic candidates. Triclosan inhibits an essential enzyme in the fatty acid biosynthetic pathway, the reduced nicotinamide adenine dinucleotide (NADH)/reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enoyl-acyl carrier protein (enoyl-ACP) reductase, or FabI. In this study, we used error-prone polymerase chain reaction (epPCR) to generate mutations in the S. aureus FabI enzyme. Instead of using an elaborate FabI enzyme activity assay that involves ACP-linked substrates to determine whether triclosan inhibits the enzyme activities of individual FabI mutants, we used an efficient and economical assay that we developed, based on thermal shift principles, to screen for triclosan binding to FabI mutants in cells. We identified four active-site mutations. More interestingly, we also identified nine triclosan-resistant mutations distant from the active site (G113V, Y123H, S166N, N220I, G227C, A230T, V241I, F252I, and H253P) but located in disparate positions in the monomer-monomer and dimer-dimer interface regions in S. aureus FabI. We suggest that these sites may serve as potential allosteric sites for designing potential therapeutic inhibitors that offer advantages in selectivity since allosteric sites are less evolutionarily conserved.