COUNTERING BACTERIAL RESISTANCE VIA INHIBITION OF TWO-COMPONENT SYSTEMS

Abstract

The emergence of bacterial resistance is a current and rapidly growing threat to human health worldwide. Each year, pathogenic bacteria exhibit new or increased resistance to a limited arsenal of existing antibiotics at a rate that greatly outpaces new antimicrobial discovery. In several species the reality of pan-resistant bacterial infections with no means of treatment has already arrived. To counter this, the production of creative, unconventional therapeutics with specific concern for eliminating or reducing resistance development is imperative. To achieve this goal, the development and application of antibiotic adjuvants - a therapeutic that enhances the effect of another treatment - offers a promising solution. By nature, successful application of an adjuvant would allow for restored efficacy of existing antimicrobials and offset the massive time and monetary investment required for developing new antibiotics. Two-component systems (TCSs) serve as ubiquitous communication modules that enable bacteria to detect and respond to various environmental stimuli by regulating cellular processes such as growth, viability, and most notably, antimicrobial resistance. Classical TCSs 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, namely the upregulation of genes that elicit bacterial defense mechanisms. TCSs are undeniably one of the most effective therapeutic targets against bacterial resistance. Given their function, the inhibition of the TCS would remove the bacteria’s ability to sense and respond to changes in its environment, i.e. the presence of antimicrobials. In laymen’s terms, these treatments effectively ‘blind’ the bacteria to the threat of the therapeutic/antibiotic, removing the ability to activate resistance mechanisms. Further, due to these therapeutics ability to render bacteria oblivious to the threat of the antibiotics, there is no increase in selective pressure. As a result, the bacteria do not develop resistance to the adjuvant compounds or increased resistance to the antibiotic. As the initiators of the signaling pathways that elicit resistance, the histidine kinases present as the ideal target within the TCS for developing antibiotic adjuvant drugs. Despite this, due to the membrane-bound nature of histidine kinases, in vitro investigations for TCSs have been predominantly limited to response regulators. This includes the development of targeted therapeutics. In this work, we counter this limitation by producing recombinant truncation mutants of the cytosolic portion of HKs that retain ATP-binding, autophosphorylation, and phosphotransfer functions. This method was initially used in A. baumanii’s PmrAB system to make a truncation of PmrB (Polymyxin resistance protein B), dubbed PmrBc. The PmrAB system is the main mechanism of resistance to colistin (polymyxin E) in Acinetobacter baumannii. This truncation mutant allowed for in vitro evaluation of potential salicylanilide histidine kinase inhibitors, previously shown to eliminate resistance in vivo. Experimental findings from kinase assays, limited proteolysis, and hydrogen-deuterium exchange (HDX) mass spectrometry enabled us to determine these compounds’ mechanism of action as well as the likely binding site on the ATP-lid of the histidine kinase’s catalytic domain. Following successful production of PmrBc, other functional cytosolic truncations were produced across multiple species for several resistance mechanisms, including Klebsiella pneumonia. While K. pneumonia also utilizes the PmrAB system to produce colistin resistance, it contains a second mechanism, PhoPQ, that is capable of upregulating resistance genes outside those of the PmrAB system. With the goal of addressing both systems, truncations of K. pneumonia PhoQ and PmrB were produced for screening and evaluation of the salicylanilide inhibitors. Notably, these results revealed variations in specificity/selectivity of the compounds between kinases, suggesting solution of structural data for the kinases will allow for structure-activity-relationship (SAR) based drug design and enable targeting of specific HKs. Beyond applications for drug development, truncation mutants also enabled expanded in vitro investigation of TCSs. The cytosolic construct of A. baumannii PmrB, was utilized to assess the effect of resistance inducing point mutations in its cognate response regulator. Results of phosphorylation assays identified discrepancies in the phosphorylation rates between point mutants, distinguishing altered phosphotransfer activity as the means of increased Polymyxin resistance within these clinical isolates. Finally, previous efforts have also been successful in targeting the response regulators within the TCS. A class of compounds called 2-aminoimidizoles (2-AIs), developed from a natural product, have shown the ability to target response regulators. Early work gave clues to their mechanism of action and binding site but were never confirmed. Our recent assessment of the effects of two novel 2-AIs on the PhoPQ system support this previously proposed mechanism of action and indicate how these compounds affect transcriptional regulation. In whole, the work presented here cohesively establishes the efficacy, binding site, and mechanism of action for 2-AI compounds in inhibiting response regulators within the PhoPQ system. In summation, this work utilizes a range of biochemical techniques to evaluate compounds aimed at inhibiting the histidine kinase and response regulator of TCSs to ameliorate antibiotic resistance. We also develop methods for producing functional cytosolic truncation mutants that counter current limitations in research of two-component systems. These works offer promising avenues in countering antibiotic resistance that simultaneously limit the potential for bacteria to develop resistance to the adjuvant compounds or increased resistance to antibiotics.