An Efficient and Economical Assay to Screen for Triclosan Binding to FabI

Abstract

Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosanresistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP + to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

Chronic Exposure to an Environmentally Relevant Triclosan Concentration Induces Persistent Triclosan Resistance but Reversible Antibiotic Tolerance in Escherichia coli

“…30 Reportedly, fabI, which catalyzes the final step in each fatty acid elongation cycle, is an important target of TCS. 31 Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis verified that fatty acid-related pathways (i.e., synthesis, metabolism, and degradation) were differentially enriched in adapted cells relative to wild-type E. coli upon TCS exposure (Figure 3a). The KEGG enrichment analysis of DEGs between E TCS and E. coli is displayed in the SI (Excel file 2).…”

“…TCS targets enoyl-acyl carrier protein reductases, blocking bacterial type II fatty acid synthesis . Reportedly, fabI , which catalyzes the final step in each fatty acid elongation cycle, is an important target of TCS . Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis verified that fatty acid-related pathways (i.e., synthesis, metabolism, and degradation) were differentially enriched in adapted cells relative to wild-type E.…”

Nonactive-Site Mutations in S. aureus FabI That Induce Triclosan Resistance

“…The gel band of the same representative cell sample, but heated to 65 °C, was hardly visible ( Figure 1 , panel A, lane 3), with an intensity of only 0.02 (or 2.2% of that at 37 °C) for this particular sample, indicating that Sa FabI in cells was hardly soluble at 65 °C, as expected. 28 The average intensity value of 13 runs at 65 °C was 0.03 ± 0.01 (or 2.6 ± 1.6% of that at 37 °C, with values ranging from 0.5 to 5.9%) ( Table 1 , WT, 0×).…”

“…Consequently, screening for triclosan-resistant mutants is challenging. Based on cellular thermal shift assay method, 27 we used the simple thermal shift assay that we developed, 28 which does not use ACP-linked substrates, to screen for triclosan-resistant mutants in this study. Since each individual protein exhibits a characteristic thermal unfolding profile in solution, the unfolded proteins at a specific elevated temperature form aggregates and are no longer soluble in the solution.…”

“…The pET-15b- Sa FabI mutant plasmid was extracted from individual colonies, and the SaFabI mutant gene was sequenced (Sanger sequencing at the University of Illinois at Chicago (UIC) Research Resources Center (RRC)) and compared with the sequence of the WT Sa FabI gene. Some of the mutated cells were randomly selected, and their gene products were purified with affinity column chromatography methods 28 for high-resolution mass spectrometry analysis by the UIC RRC to confirm the mutation identities.…”