Evaluating the Link between Efflux Pump Expression and Motility Phenotypes in <i>Pseudomonas aeruginosa</i> Treated with Virulence Inhibitors

Antibiotic resistance continues to rise as a global health threat. Novel antivirulence strategies diminish the drive for evolutionary pressure but still hinder a pathogen's ability to infect a host. Treatment of the highly virulent <i>Pseudomonas aeruginosa</i> strain PA14 with virulence inhibitors (R-2 and R-6) elicited widely varying transcriptional profiles. Of interest, the expression of a family of resistance-nodulation-division (RND) efflux pumps implicated in the intrinsic drug resistance of <i>P. aeruginosa</i> was significantly altered by R-2 and R-6 treatment. While structurally similar, these inhibitors caused differential expression of various RND efflux pumps within the Mex family─the R-2 treatment stimulated the expression of <i>mexEF-oprN</i>, while the R-6 treatment led to increased <i>mexAB-oprM</i> expression. Further expansion into a small library of virulence inhibitors revealed chemical motifs that trigger increases in the level of RND efflux pump expression. Additionally, activation of these efflux pumps suggests a low accumulation of virulence inhibitors in WT PA14. Treatment of an efflux pump-deficient strain with R-2 or R-6 resulted in inhibition of several virulence factors; for example, R-2 was found to abolish swimming motility. Collectively, treatment with either R-2 or R-6 gives rise to a convoluted transcriptomic response confounded by the impact of efflux pump expression on the system. However, understanding the moieties that lead to high expression of the efflux pumps enables the further rational design of novel virulence inhibitors that do not cause RND efflux pump activation.