Evaluation of ExoU inhibitors in a Pseudomonas aeruginosa scratch infection assay

Abstract

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system of P. aeruginosa delivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy in P. aeruginosa infections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activity in vitro. A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU from P. aeruginosa. Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 M. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producing P. aeruginosa.