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

铜绿假单胞菌（Pseudomonas aeruginosa）是一种引起多种感染的细菌，包括角膜（眼睛）、肺部和血液感染，在世界范围内造成严重的残疾和死亡。最近耐抗生素假单胞菌的激增促使世界卫生组织建议必须作为“高度优先”开发新的治疗方法。许多病人的康复情况仍然很差，即使他们的病情可以用抗生素治疗。对于那些角膜（眼睛前部的透明窗口）感染的人来说，严重的损伤和疤痕可能导致失明。在结果不佳的假单胞菌感染中，已经确定细菌将一种称为外毒素U （ExoU）的毒素注入人体细胞。ExoU会迅速杀死细胞，包括用来对抗感染的免疫细胞。细菌逃避免疫系统继续繁殖并造成损害。抑制ExoU可能为降低该细菌引起的严重程度、致残和死亡提供新的治疗方法。我们已经确定了25种有前景的药物可以特异性抑制ExoU。在角膜感染的试管模型中，这些药物减少了损伤，并显示出保护作用，帮助细胞愈合。即使在非常低的浓度下也会发生这种情况。我们的数据表明，它们可以与抗生素结合使用来改善这些结果。在人体试验之前需要进行重要的研究。这包括测试不同的浓度和组合，评估它们到达感染部位的程度，并在临床相关的动物模型中进行测试。管理方案也需要，包括课程的强度、频率和长度。证明它们对其他器官（如肺）的感染有效，将证明它们可以被开发出来治疗并使更多的患者受益。我的奖学金项目将以我们最初的工作为基础，有以下目的和目标：1)我将在划痕和感染细胞模型中评估ExoU抑制药物，主要是在眼睛细胞中，但也在肺细胞中，看看它们是否也能在这些细胞中起作用。在使用和不使用抗生素的情况下，将测试不同的浓度和组合。结果将展示其潜力，并有助于制定治疗方案。我将测量到达感染部位的ExoU抑制药物的浓度。我将使用一个模型，该模型将捐赠的人类角膜附着在眼睛前房（含有液体的前部）的玻璃人工模型上。这种药物，作为眼药水，将被应用于表面，并在不同的时间点对液体进行取样。将测量这些样品中的药物浓度。这是一个重要的信息，它将有助于制定治疗方案，并推动药物进行人体试验。3)我将在更高级的感染模型中评估ExoU抑制药物。猪角膜（食品工业的副产品）将被假单胞菌感染，并使用目标1和目标2中制定的方案用ExoU抑制剂治疗。这将有助于确定最有希望的药物和完善治疗方案，将在进一步的实验中使用。进行本实验将大大减少后续实验所需的动物数量。4)当我确定了两种最佳药物和方案后，我将在角膜感染的小鼠模型中进一步评估它们。这项工作将与我在美国的合作者一起进行，他们有一个道德上认可的模型和专业知识。这些实验将提供必要的数据，包括安全性数据，我们将需要这些数据进行人体临床试验。我的项目将显著发展将这些ExoU抑制药物推向人体试验所需的知识。最终目标是，这些药物将用于治疗假单胞菌感染患者，以改善他们的康复和总体结果。

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.