Active and Non-Active Site Mutations in Staphylococcus aureus FabI Responsible for Triclosan Resistance

Up to 30% of the world population is colonized with nasal Staphylococcus aureus and nasal colonization can cause opportunistic and sometimes life-threatening infections. Triclosan is an antimicrobial agent/biocide, originally used in hospital settings as a disinfectant to remove bacterial colonies, including S. aureus, and was incorporated into a variety of consumer products. Triclosan inhibits the enoyl-acyl carrier protein reductase, or FabI, an essential enzyme in the fatty acid biosynthetic pathway in many pathogens including S. aureus. Bacterial cells mutation rate is roughly 0.003 mutations per genome per cell generation; mutations continuously occur naturally. The extensive use of triclosan selects S. aureus enzyme FabI mutants that do not bind triclosan. A simple method to determine which mutant does not bind triclosan is important. We have developed an efficient and economical method to test for triclosan binding to mutants, using a thermal shift method. We applied this method to S. aureus cells with FabI mutants generated randomly in laboratory. We showed that four mutants with mutations in active site did not bind triclosan as expected. We also identified nine mutations that are not located at the active site, but affect triclosan binding. We found that the mutations were in disparate positions in the monomer-monomer and dimer-dimer interface regions. We suggest that these sites may serve as potential allosteric sites that can be targeted by potential therapeutics to inhibit the FabI from S. aureus. In addition, we have observed cellular thermal shift assay results vary for certain mutants when conducted in intact cells compared to lysate conditions. We attribute these differences to quinary interactions that are present in the cell, but mostly abolished in the lysate.