How Acinetobacter baumanniiControls Antibiotic Resistance through Phosphorylation of the BfmR Global Regulator.

There is an evolving threat to our healthcare system-the rise of antibiotic-resistant bacteria, such as Acinetobacter baumannii, and the depletion of our few useful antibiotics. These bacteria have developed an ability to fight off increasing numbers of antibiotics used to treat hospitalized patients. To combat these infections, new solutions must be discovered to target the bacteria's ability to resist drugs and restore the efficacy of our arsenal of antibiotics. One possible therapeutic target is the BfmRS two-protein signaling system, which activates many genes that determine antibiotic resistance and virulence in A. baumannii. The transcription factor, BfmR, is key to this gene regulation pathway. How the activity of BfmR is controlled is unknown. We hypothesize that a phosphorylation signal activates BfmR, allowing it to form an oligomeric state that binds to and enhances expression of target genes. To test this hypothesis, we developed in vitrophosphorylation conditions using purified BfmR and phosphoramidate, a small molecule phosphodonor. We used phosphate-affinity polyacrylamide gel electrophoresis to determine that phosphorylation of the purified protein was highly efficient. Size exclusion chromatography analysis of BfmR using high-performance liquid chromatography (HPLC) was consistent with dimerization of the protein upon phosphorylation. Next steps will include evaluating the binding affinity of the phosphorylated protein to a series of target DNA sequences associated with resistance and virulence genes, chosen from chromatin immunoprecipitation sequencing (ChiP-Seq) data showing conserved response elements. We will employ electrophoretic mobility shift assays (EMSAs) to analyze how phosphorylation affects BfmR binding to these sequences. The information we uncover on the regulatory mechanisms of BfmR can be used to guide the development of inhibitors that counteract A. baumannii's resistance to essential antibiotics.