Two-Component System Sensor Kinase Inhibitors Target the ATP-Lid of PmrB to Disrupt Colistin Resistance in Acinetobacter baumannii

Two-component systems serve as ubiquitous communication modules that enable bacteria to detect and respond to various stimuli by regulating cellular processes such as growth, viability, and, most notably, antimicrobial resistance. Classical two-component systems consist of two proteins: an initial membrane-bound sensor histidine kinase and a DNA-binding response regulator that induces the appropriate response within the cell. Numerous studies have implicated the PmrAB two-component system in facilitating resistance to the last-resort antibiotic polymyxin E (colistin) in Acinetobacter baumannii. As initiators of the signaling pathways that elicit resistance, histidine kinases present ideal targets for developing antibiotic adjuvant drugs. Despite this, due to the membrane-bound nature of the histidine kinase PmrB, in vitro studies on PmrAB have been predominantly limited to the response regulator PmrA. In this work, we counter these limitations by producing a recombinant truncation of the cytosolic portion of PmrB (PmrBc) that retains its ATP binding, autophosphorylation, and phosphotransfer functions. Subsequently, in vivo phosphorylation assays using this protein construct allowed for the evaluation of five compounds (IMD-0354, NDM-265, NDM-455, NDM-463, and NDM-497) that act as PmrBc inhibitors capable of preventing autophosphorylation and phosphotransfer independently. These compounds have been shown to eliminate colistin resistance in vivo. Finally, these results, paired with mass spectrometry and limited proteolysis investigations, enabled us to determine the mechanism of action of these compounds as well as their likely binding site on the ATP-lid of PmrB.