A dual-specificity inhibitor targets polyphosphate kinase 1 and 2 enzymes to attenuate virulence of pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections, which are becoming increasingly difficult to treat due to antibiotic resistance. Polyphosphate (polyP) plays a key role in P. aeruginosa viru-lence, stress response, and antibiotic tolerance, suggesting an attractive drug target. Here, we show that the small molecule gallein disrupts polyphosphate homeostasis by inhibiting all members of both polyphosphate kinase (PPK) families (PPK1 and PPK2) encoded by P. aeruginosa, demonstrating dual-specificity PPK inhibition for the first time. Inhibitor treatment phenocopied ppk deletion to reduce cellular polyP accumulation and attenuate biofilm formation, motility, and pyoverdine and pyocya-nin production. Most importantly, gallein attenuated P. aeruginosa virulence in a Caenorhabditis elegans infection model and synergized with antibiotics while exhibit-ing negligible toxicity toward the nematodes or HEK293T cells, suggesting our discovery of dual-specificity PPK inhibitors as a promising starting point for the devel-opment of new antivirulence therapeutics. IMPORTANCE Many priority bacterial pathogens such as P. aeruginosa encode both PPK1 and PPK2 enzymes to maintain polyphosphate homeostasis. While PPK1 and PPK2 have distinct structures and catalytic mechanisms, they are both capable of synthesizing and consuming polyphosphate; thus, PPK2 enzymes can compensate for the loss of PPK1 and vice versa. In this study, we identified the small molecule gallein as a dual-specificity inhibitor of both PPK1 and PPK2 enzyme families in P. aeruginosa. Inhibitor treatment reduced cellular polyP in wild-type (WT), Δppk1, and Δppk2 strains to levels that were on par with the Δppk1 Δppk2A Δppk2B Δppk2C knockout control. Treatment also attenuated biofilm formation, motility, toxin pro-duction, and virulence to a similar extent, thereby elucidating a hitherto-undocu-mented role of PPK2 enzymes in P. aeruginosa virulence phenotypes. This work therefore establishes PPK2s, in addition to PPK1, as valuable drug targets in P. aeru-ginosa and provides a favorable starting molecule for future inhibitor design efforts.