Loss of RND-Type Multidrug Efflux Pumps Triggers Iron Starvation and Lipid A Modifications in Pseudomonas aeruginosa.

Transporters belonging to the resistance-nodulation-division (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The numbers of genes encoding RND transporters per genome vary from 1 to 16 and correlate with the environmental versatilities of bacterial species. Pseudomonas aeruginosa strain PAO1, a ubiquitous nosocomial pathogen, possesses 12 RND pumps, which are implicated in the development of clinical multidrug resistance and known to contribute to virulence, quorum sensing, and many other physiological functions. In this study, we analyzed how P. aeruginosa's physiology adapts to a lack of RND-mediated efflux activities. A combination of transcriptomics, metabolomics, genetic, and analytical approaches showed that the P. aeruginosa PΔ6 strain, lacking the six best-characterized RND pumps, activates a specific adaptation response that involves significant changes in the abundance and activities of several transport system, quorum sensing, iron acquisition, and lipid A modification pathways. Our results demonstrate that these cells accumulate large quantities of Pseudomonas quinolone signals (PQS), which triggers iron starvation and activation of siderophore biosynthesis and acquisition pathways. The accumulation of iron in turn activates lipid A modification and membrane protection pathways. A transcriptionally regulated RND pump, MuxABC-OpmB, contributes to these transformations by controlling the concentration of coumarins. Our results suggest that these changes reduce the permeability barrier of the outer membrane and are needed to protect the cell envelope of efflux-deficient P. aeruginosa.

Identification and Structure-Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli.

In Gram-negative bacteria, efflux pumps are able to prevent effective cellular concentrations from being achieved for a number of antibiotics. Small molecule adjuvants that act as efflux pump inhibitors (EPIs) have the potential to reinvigorate existing antibiotics that are currently ineffective due to efflux mechanisms. Through a combination of rigorous experimental screening and in silico virtual screening, we recently identified novel classes of EPIs that interact with the membrane fusion protein AcrA, a critical component of the AcrAB-TolC efflux pump in Escherichia coli. Herein, we present initial optimization efforts and structure-activity relationships around one of those previously described hits, NSC 60339 (1). From these efforts we identified two compounds, SLUPP-225 (17h) and SLUPP-417 (17o), which demonstrate favorable properties as potential EPIs in E. coli cells including the ability to penetrate the outer membrane, improved inhibition of efflux relative to 1, and potentiation of the activity of novobiocin and erythromycin.