Antibiotic Adjuvant Activity Revealed in a Photoaffinity Approach to Determine the Molecular Target of Antipyocyanin Compounds

Abstract

Infections with Pseudomonas aeruginosa are a looming threat to public health. New treatment strategies are needed to combat this pathogen, for example, by blocking the production of virulence factors like pyocyanin. A photoaffinity analogue of an antipyocyanin compound was developed to interrogate the inhibitor's molecular mechanism of action. While we sought to develop antivirulence inhibitors, the proteomics results suggested that the compounds had antibiotic adjuvant activity. Unexpectedly, we found that these compounds amplify the bactericidal activity of colistin, a well-characterized antibiotic, suggesting they may represent a first-in-class antibiotic adjuvant therapy. Analogues have the potential not only to widen the therapeutic index of cationic antimicrobial peptides like colistin, but also to be effective against colistin-resistant strains, strengthening our arsenal to combat P. aeruginosa infections.

Keywords: ArnA; PA14_30820; Pseudomonas aeruginosa; adjuvant; colistin.

Figure 1.. Phenazine production.

(A) Biosynthesis of pyocyanin in P. aeruginosa. Adapted from references 16-19. (B) Production of phenazines upon treatment with compound 1. The production of PCA and pyocyanin (PYO) in wild-type P. aeruginosa PA14 (WT PA) is inhibited by compound 1, while heterologous PCA production in the recombinant E. coli (EC) strains are not inhibited. WT P. aeruginosa PA14 and the phz-containing E. coli strains were grown in MOPS-LB in the presence of DMSO or 1 (100 μM). PCA and PYO levels in cell-free supernatants were quantified by HPLC. Error bars represent the standard deviation of the mean of 3 replicates across 1 day (EC) or 15 replicates across 5 days (PA). Statistical significance was determined by t tests comparing the DMSO and compound 1 treatments within each strain. ****, p < 0.0001. (C) Inhibitor 1 blocks 99% production of pyocyanin at 100 μM in WT P. aeruginosa PA14 by the UV/Vis assay. Activity data from reference 9.

Figure 2.. PAL target.

(A) Design of PAL target based on structure-activity relationships. (B) Synthesis of PAL 5. Inhibition activity from the UV/Vis assay. Activity data from reference 9.

Figure 3.. Compound 2 increases susceptibility to colistin.

(A) P. aeruginosa PA14 was treated for 17 h with increasing amounts of colistin in the presence of no added inhibitor (DMSO, circles) or 50 μM inhibitor 2 (triangles). (B) P. aeruginosa PA14 was treated with increasing amounts of inhibitor 2 in the presence of no added colistin (water, circles) or 0.375 μg/mL colistin (triangles). (A-B) Cell viability was tracked by OD600. Error bars represent the standard deviation of the mean of three replicates.