Topical formulation of repurposed FDA-approved compounds inhibits Pseudomonas aeruginosa ExoU and improves corneal infection outcomes.

Microbial keratitis caused by Exotoxin U (ExoU)-producing strains of Pseudomonas aeruginosa is associated with poor clinical outcomes and reduced responsiveness to antimicrobial therapy. ExoU, a phospholipase, is secreted directly into host cells, causing their lysis. A screen of 3,034 FDA-approved compounds identified zinc pyrithione (Zp), bismuth subcitrate (Bis), and polymyxin B (Pol) as lead inhibitors of ExoU selectively inhibiting it without affecting human PLA2s or bacterial viability. The compounds have distinct inhibitory mechanisms: Zp disrupted ExoU oligomerization and protein stability; Bis impaired phosphatidylinositol 4,5-bisphosphate-dependent membrane association; Pol directly inhibited catalysis via its lipid-peptide architecture. Compound combinations enhanced ExoU inhibition in vitro to nanomolar concentrations. In mammalian cells, Bis and Pol promoted lysosomal trafficking and degradation of ExoU. A high content screen of 52 P. aeruginosa keratitis isolates demonstrated the broad efficacy of these inhibitors in exoU⁺ strains. Therapeutic efficacy was evaluated in ex vivo porcine corneas, Galleria mellonella, and in vivo mouse keratitis models. Topical delivery of ExoU inhibitors, particularly in combination, significantly reduced corneal opacity, ulceration, and stromal damage in porcine corneas without affecting bacterial load. In Galleria, compound combinations significantly maintained larval health, improved larval survival and delayed mortality. In a mouse eye infection model, combinatorial treatment reduced disease severity and preserved tissue viability without altering bacterial burden. These findings validate ExoU as a druggable virulence factor and support the repurposing of these compounds as an antivirulence strategy for the treatment of P. aeruginosa infections in human beings and in veterinary medicine.