Targeting Exotoxin U with repurposed drugs to improve the clinical outcomes of Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa (Pseudomonas) is a bacteria which causes a wide variety of infections, including corneal (eye), lung and blood, resulting in significant disability and death worldwide. A recent surge in antibiotic-resistant Pseudomonas has prompted the World Health Organisation to advise new treatments must be developed as a 'high priority'. Recovery for many patients remains very poor even if their condition is treatable with antibiotics. For those with corneal (transparent window at the front the eye) infections, significant damage and scarring can lead to blindness.
In Pseudomonas infections with poor outcomes it has been established that the bacteria inject a toxin, called Exotoxin U (ExoU), into human cells. ExoU rapidly kills the cells including immune cells sent to fight the infection. The bacteria evade the immune system to continue multiplying and causing damage. Inhibiting ExoU could provide a new treatment to reduce the severity, disability and death caused by this bacteria.
We have identified 25 promising drugs which specifically inhibit ExoU. In test-tube models of corneal infections, the drugs reduce damage and show protective effects which help the cells heal. This occurs even at very low concentrations. Our data suggests they can be combined with antibiotics to improve these results.
Significant research is required prior to human trials. This includes testing different concentrations, combinations, assessing how well they get to the site of infection, and testing them in clinically-relevant animal models. Protocols for administration are also required, including strength, frequency and length of the course. Demonstrating they work for infections in other organs, such as the lung, would prove they could be developed to treat and benefit more patients.
My fellowship project will build on our initial work, with the following aims and objectives:
1) I will evaluate the ExoU inhibiting drugs in scratch and infection cell models, primarily in eye cells, but also lung cells to see if they will work in these too. Different concentrations and combinations will be tested, with and without antibiotics. Results will demonstrate their potential and help develop the treatment protocols.
2) I will measure the concentration of the ExoU inhibiting drugs that get to the site of infection. I will utilise a model which uses a donor human cornea attached to a glass artificial model of the anterior chamber of the eye (the front part which contains fluid). The drug, as an eye-drop, will be applied to the surface, and samples of the fluid within will be taken at different time-points. The concentration of drug in these samples will be measured. This is important information which will aid the development of the treatment protocols and advance the drugs towards human trials.
3) I will evaluate the ExoU inhibiting drugs in a more advanced infection model. Pig corneas (by-products of the food industry) will be infected with Pseudomonas and treated with the ExoU inhibitors using the protocols developed in objectives 1 and 2. This will help identify the most promising drugs and refine the treatment protocol which will be used in further experiments. Conducting this experiment will significantly reduce the number of animals required in subsequent experiments.
4) When I have identified the two best drugs and protocols, I will evaluate these further in a mouse model of corneal infection. This work will be conducted with my collaborators in the United States who have an ethically approved model and the expertise. These experiments will provide the necessary data, including the safety data, which we will require to proceed to human clinical trials.
My project will significantly develop the knowledge required to progress these ExoU inhibiting drugs towards human trials. The ultimate aim is that the drugs will be used to treat patients with Pseudomonas infections to improve their recovery and overall outcome.

Exotoxin U (ExoU) is significantly linked to poor outcomes of Pseudomonas aeruginosa infections. It is a phospholipase injected into host cells, which causes rapid destruction of tissue and evasion of the immune system. We hypothesis targeting ExoU with drugs could provide a novel therapy that improves clinical outcomes.
We have identified 25 promising drugs which inhibit ExoU. In vitro cell models demonstrate they reduced cell lysis and promote healing. A synergistic effect is also shown with some conventional antimicrobials.
This project aims to establish the efficacy and pharmacokinetics of ExoU inhibitors, specifically their ability to reduce tissue damage in cell, ex vivo and in vivo models.
Utilising scratch and infection models of human corneal (HCE-t) and human lung epithelial (A549) cell lines, I will determine which compounds reduce the cellular damage associated with ExoU, with and without additional antimicrobials. Cellular responses will be quantified by lactate dehydrogenase, trypan blue uptake and propidium iodide uptake assays, alongside live-cell and immunoflourescence imaging.
Human donor cornea attached to an artificial anterior eye chamber model will be used to demonstrate the concentration of drugs which penetrate through the cornea. Concentrations will be estimated using a phospholipase assay compared with a standard curve and a select number will be quantified and validated using liquid chromatography-mass spectrometry.
Treatment protocols for future work will be established using an ex vivo porcine corneal infection model. Different concentrations and combinations of drugs will be tested, with and without antibiotics. Over 72 hours, clinical scoring, colony forming units (CFU) counts and histology will be analysed.
Finally, an ethically approved in vivo murine model of P. aeruginosa corneal infection will be used to establish safety and efficacy data using clinical scoring, CFUs and histology.