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

Abstract

Antibiotic resistance continues to rise as a global health threat. Novel anti-virulence strategies diminish the drive for evolutionary pressure, but still hinder a pathogens ability to infect a host. Treatment of the highly virulent Pseudomonas aeruginosa strain PA14 with virulence inhibitors (R-2 and R-6) 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 R-2 and R-6 treatment. While structurally similar, these inhibitors caused differential expression of various RND efflux pumps within the Mex family--R-2 treatment stimulated expression of mexEF-oprN while R-6 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 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 further rational design of novel virulence inhibitors that do not cause RND efflux pump activation. SummaryAntibiotic resistance continues to rise across the world. The continued failure of antibiotics to treat infectious disease clearly demonstrates the need for new therapeutics. Small molecule inhibitors of virulence, or the ability of a bacteria to establish and maintain an infection, have showed promise in this area. By reducing virulence, the host immune system can clear the infection on its own and there is less selective pressure on the bacteria, reducing the emergence of resistance. Our work demonstrates a clear link between virulence inhibitors and efflux pump expression. Efflux pumps are critical for bacteria to extrude toxic compounds from their interior. Interestingly, the changes in efflux pump expression can be quite large even with similar molecular structures. Additionally, the repression of efflux expression caused by our virulence inhibitors results in synergy with current antibiotics. This work will allow the future design of virulence inhibitors that do not activate efflux pump expression and therefore, enable high intracellular concentrations.