Blocking Cationic Antimicrobial Peptide-Resistance in Pseudomonas aeruginosa

项目总结/摘要 抗药性细菌菌株的感染对国家的公共卫生构成了迫在眉睫的威胁。的 在COVID-19时代，这个问题尤其紧迫，在一半的人口中发现了继发性感染， 没有存活下来的住院患者。对继发性感染的身份进行更多的研究， 目前的疫情仍需进行，但在SARS冠状病毒疫情中，革兰氏阴性菌 是医院感染的最常见来源。革兰氏阴性菌，如假单胞菌 铜绿假单胞菌几乎没有有效的治疗方法，使得任何耐药菌株特别难以治疗。 抗微生物肽（CAP）如粘菌素目前用作治疗感染的最后一线疗法， 多药耐药菌株，然而，越来越多地遇到粘菌素耐药菌株。铜绿假单胞菌 通过修饰其外膜的脂质以减少净负电荷而变得对CAP具有抗性。 在带正电的CAP和带负电的CAP之间没有有利的静电相互作用的情况下， 当细菌外膜带电时，抗生素不能进入并杀死细菌。我们的目标是开发 并表征细菌中CAP抗性途径的抑制剂。如果外膜仍然存在 负电荷，联合呼吁程序应继续工作。所述抑制剂可用作免疫抑制剂中的佐剂。 联合治疗沿着CAP以治疗耐药感染。 在第一个资助期内，我们确定了一套作为抗生素佐剂的化合物， 对粘菌素敏感。在目前的工作中，我们建议对联合用药的疗效进行表征 治疗（目标1）。我们将研究对生物膜、耐药菌株和耐药菌株的活性- 诱导条件。还将研究杀灭的动力学和抗性的发展。我们目前 假设是抗生素佐剂通过抑制ArnA起作用，ArnA是一种关键的双功能生物合成酶， 脂质修饰途径。我们将研究佐剂对ArnA介导的抗性的影响 通过质谱法分析脂质和在动力学中与ArnA的直接相互作用， 结合实验（目标2）。所提出的工作将验证该组合的进一步发展 在临床上的应用。分子靶点的确认也将指导甚至是 更有效的抑制剂在未来的工作。破译分子靶点也可以让我们扩展 治疗其他革兰氏阴性菌，如碳青霉烯耐药肠杆菌科和耐药 沙门氏菌作为一个长期目标。这种一流的抑制剂有可能扩大 粘菌素，最大限度地减少药物的严重副作用，以及使粘菌素耐药的治疗 菌株，确保救生药物的持续有效性。

Project Summary/Abstract Infections with antibiotic-resistant strains of bacteria are a looming public health threat to the nation. The problem is especially pressing in the era of COVID-19, where secondary infections were identified in half of hospitalized patients who did not survive. More research on the identity of the secondary infections in the current outbreak still needs to be conducted, but in the SARS coronavirus outbreak, gram-negative bacteria were the most common source of the hospital-acquired infections. Gram-negative bacteria like Pseudomonas aeruginosa have few effective treatments, making any resistant strains particularly difficult to treat. Cationic antimicrobial peptides (CAPs) like colistin are currently used as last line therapies to treat infections with multidrug-resistant strains, however, colistin-resistant strains are increasingly encountered. P. aeruginosa becomes resistant to CAPs by modifying the lipids of its outer membrane to reduce the net negative charge. Without the favorable electrostatic interaction between the positively-charged CAP and the negatively- charged bacterial outer membrane, the antibiotic cannot enter and kill the bacterium. Our goal is to develop and characterize inhibitors of the CAP-resistance pathway in the bacterium. If the outer membrane remains negatively charged, the CAP should continue to work. The inhibitors could be used as an adjuvant in a combination therapy along with a CAP to treat resistant infections. In the first funding period, we identified a suite of compounds that act as antibiotic adjuvants, potentiating susceptibility to colistin. In the current work, we propose to characterize the efficacy of the combination treatment (Aim 1). We will investigate activity toward biofilms, resistant strains, and under resistance- inducing conditions. The kinetics of killing and the development of resistance will also be studied. Our current hypothesis is that the antibiotic adjuvant acts by inhibition of ArnA, a key bifunctional biosynthetic enzyme of the lipid modification pathway. We will study the adjuvant’s effects on the ArnA-mediated resistance pathway through analysis of the lipids by mass spectrometry and direct interactions with ArnA in kinetics and binding experiments (Aim 2). The proposed work will validate the further development of the combination therapy towards use in the clinic. Confirmation of the molecular target will also guide the optimization of even more potent inhibitors in future work. Deciphering the molecular target could also allow us to extend the therapy to other gram-negative bacteria like carbapenem-resistant Enterobacteriaceae and drug-resistant Salmonella as a long-term goal. The first-in-class inhibitor has the potential to widen the therapeutic index of colistin, minimizing the drug’s serious side effects, as well as to enable the treatment of colistin-resistant strains, ensuring the continued efficacy of the life-saving drug.