The Role of Response Regulators in Gram-Negative Bacterial Resistance

Abstract

Since their beginning, antibiotics have been hailed as a miracle of modern medicine. And while their discovery brought about exciting advances across a multitude of industries, the ubiquity of their use has paved the path leading towards the current resistance crisis and the dawn of a post-antibiotic era. As resistant bacteria have emerged steadily over time, updated methods of treating infections have developed. From double treatments using two antibiotics or recycling older antibiotics, these changes have come about in hopes to delay resistance and prolong the lifespan of the antibiotics currently in use. Included in the older classes of antibiotics is polymyxins, their usage gone to wayside due to their innate nephro and neuro-toxic characteristics. The use of polymyxins has now been reconsidered to overcome highly resistant pathogens typically found in healthcare settings. And while the side effects of polymyxin treatment can be deleterious it is a more preferable outcome than succumbing to a fatal infection. Nosocomial infection have become more and more prevalent, threatening patients within ICUs and under long term care. Some of the major contributors of the most concerning nosocomial infections are the E.S.K.A.P.E. pathogens, these are (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Named for their ability to escape treatment, they are notorious for causing dangerous infections and rapidly developing resistance characteristics. Globally recognized institutions such as the World Health Organization have declared a state of emergency and have issued a call to action to academic institutions, pharmaceutical developers, and government agencies to come together to prevent the reality of a world overrun with untreatable infections. In response, there has been extensive work done to understand the variety and nuance of resistance mechanisms. Additionally, strides have been made towards newer methods of ameliorating resistance through the use of small molecule antibiotic adjuvants. Adjuvants work in tandem with drugs to improve their efficacy. The following body of work explores the mechanisms and application of two such adjuvants, 2-aminoimidazole compounds and several salicylanilide kinase inhibitors. Both of these compounds target separate components of systems found in most bacteria and are responsible for regulating the expression of resistance inducing genes. Two-component systems (TCSs) are present in most bacteria, playing a crucial role in how they sense and respond to environmental signals. TCSs have been an attractive drug target since their roles in a wide variety of cellular processes, such as nutrient uptake, motility and virulence, have been elucidated. Conveniently they appear to have no mammalian homolog, making then an ideal candidate for intervention. As their name would suggest, TCSs have two distinct components, a membrane-bound sensor kinase and a response regulator that is responsible for directly binding DNA and altering expression levels. Working as a unit, the histidine kinase responds to environmental signals by autophosphorylating and transferring the phosphoryl group to the response regulator to activate its regulatory function. While not every TCS works in this way, this is a typical example and is the most common mechanism. The TCS responsible for polymyxin resistance in species such as K. pneumoniae, A. baumannii, E. coli and numerous others is the PmrAB system. The histidine kinase, PmrB, and its cognate response regulator, PmrA, have been implicated as the mechanism responsible for inducing changes to the outer membrane of the aforementioned bacteria, these changes alter the normal charge of the outer membrane. Polymyxins are cationic and in susceptible populations of bacteria have an affinity for the negatively charged outer membrane. However, due to the PmrAB system resistant strains are able to decorate lipid-A molecules located on the outer membrane with functional groups that change the charge of the membrane as whole. The cross talk between these two molecules is subtle but complex. We have found through investigations of point mutations in highly resistant strains of A. baumannii that a single amino acid substitution can alter this signaling cascade in profound ways to increase the bacteria’s ability to survive treatments. These alterations were shown to affect PmrA’s ability to accept the phosphoryl group from PmrB in one of two ways, either by altering regions necessary for kinase recognition or perturbing the binding pocket. And while we originally hypothesized the point mutations would exert a noticeable effect of DNA-binding affinity we found that the mutants were slower to activate, lessening the energy exerted by the cell and prolonging the lifetime of the activation signal. The goal of developing small molecule adjuvants to target the PmrAB system is to interrupt the system on both sides. IMD-0354 has shown an ability to bind and inhibit the proper functioning of sensor kinase, PmrB, while 2-aminoimidazole compounds bind and inhibit the response regulator in a similar fashion. Both compounds have a minimum inhibitory concentration (MIC) lowering effect, however because polymyxins are highly toxic our goal evolved into bringing down the necessary concentration of polymyxin even lower. We performed minimum inhibitory concentration experiments to test if these two compounds can be used congruently with colistin to break the resistance mechanisms and make bacteria susceptible to treatment once again. Our endeavor was ultimately successful, bringing down the colistin MIC in resistant A. baumannii strain AB4106 to 10 µg/mL and under, a 200-fold decrease. Our goal is to apply this method to other TCSs and displace other organisms from their status as resistant pathogens.