Battling Antibiotic Resistance by Investigation of Acinetobacter baumannii Enoyl Acyl Carrier Protein Reductase

Bacterial resistance to currently available antibiotics is becoming a major public health concern. Acinetobacter baumannii is a Gram-negative bacterial species that ranks among the top antibiotic resistant pathogens. It infects compromised immune systems in humans and is a major cause of hospital infections. Narrow spectrum antibiotics need to be developed to specifically target and eliminate A. baumannii. An effective way to eliminate bacteria is by inhibiting the synthesis of fatty acids needed to build the cell membrane necessary for bacterial survival. Since humans use the fatty acid biosynthesis I pathway while bacteria use the fatty acid biosynthesis II pathway, it is safe to inhibit proteins in the fatty acid biosynthesis II pathway without affecting humans. Our therapeutic target is the enoyl acyl carrier protein reductase (FabI), which catalyzes the final step of the elongation cycle in the fatty acid biosynthesis II pathway. FabI is needed to reduce the double bond of the fatty acid being elongated in the pathway. Inhibition of FabI in A. baumannii will prevent the bacteria from generating fatty acids and lead to their demise. Here, the purification of soluble and active Acinetobacter baumannii enoyl acyl carrier protein reductase (abFabI) and initial rate kinetics of abFabI with inhibitors are described. Various chromatography methods were used to purify abFabI, and NADH consumption assays were performed at 340 nm to monitor abFabI activity. A shorter sequence (abFabI-a) and a longer sequence (abFabI-b), both designated abFabI in the literature, are also compared here, with abFabI-b including an N-terminal addition that we postulate to be a result of a sequencing error. abFabI-a was found to be homotetrameric and active while abFabI-b was misfolded and inactive. The purified active abFabI-a was used to determine IC50 values for known FabI inhibitors triclosan, four examples of compound from the diazaborine class, and isoniazid. Triclosan inhibited abFabI with an IC50 value of 0.486 μM, which was comparable to the IC50 values of triclosan on FabI homologs. Diazaborines 14b and 35b inhibited abFabI with IC50 values of 5.81 and 173 μM, respectively. In contrast, diazaborines 18c and 39 and isoniazid were not able to inhibit abFabI. Successful abFabI inhibition by triclosan and diazaborines 14b and 35b enables future development of narrow spectrum antibiotics by using these compounds for structure-based design.