Evaluating the Link Between Efflux Pump Expression and Motility Phenotypes in Pseudomonas aeruginosa Treated with Virulence Inhibitors

Antibiotic resistance continues to rise as a global health threat. Novel anti-virulence strategies diminish the drive for evolutionary pressure, but still hinder a pathogen's ability to infect a host. Treatment of the highly virulent Pseudomonas aeruginosa strain PA14 with virulence inhibitors (R2 and R6) elicited widely varying transcriptional profiles. Of interest, expression of a family of resistance-nodulation-division (RND) efflux pumps implicated in the intrinsic drug resistance of P. aeruginosa, was significantly altered by R2 and R6 treatment. While structurally similar, these inhibitors caused differential expression of various RND efflux pumps within the Mex family, R2 treatment stimulated expression of mexEF-oprN while R6 treatment led to increased mexAB-oprM expression. Further expansion into a small library of virulence inhibitors revealed chemical motifs that trigger increases in RND efflux pump expression. Additionally, activation of these efflux pumps suggests low accumulation of virulence inhibitors in WT PA14. Treatment of an efflux pump-deficient strain with R2 or R6 resulted in inhibition of several virulence factors, for example R2 was found to abolish swimming motility. Collectively, treatment with either R2 or R6 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 further rational design of novel virulence inhibitors that do not cause RND efflux pump activation.