Blocking Cationic Antimicrobial Peptide-Resistance in Pseudomonas aeruginosa

项目摘要/摘要 感染具有抗药性的细菌菌株对美国的公共健康构成了迫在眉睫的威胁。这个 在新冠肺炎时代，这个问题尤为紧迫，在这个时代，有一半的人被发现存在继发感染 住院的病人没有幸存下来。更多关于艾滋病继发感染身份的研究 目前的暴发仍需进行，但在SARS冠状病毒暴发中，革兰氏阴性菌 是医院获得性感染的最常见来源。像假单胞菌这样的革兰氏阴性细菌 铜绿假单胞菌几乎没有有效的治疗方法，这使得任何耐药菌株都特别难治疗。阳离子 像粘菌素这样的抗菌肽(CAP)目前被用作治疗感染的最后一线疗法 然而，多重耐药菌株越来越多地遇到粘菌素耐药菌株。铜绿假单胞菌 通过改变其外膜的脂类来减少净负电荷，从而对帽子产生抵抗力。 如果没有带正电的CAP和带负电的CAP之间良好的静电相互作用， 带电的细菌外膜，抗生素不能进入并杀死细菌。我们的目标是发展 以及鉴定细菌中CAP耐药途径的抑制剂。如果外膜残留 在负电荷的情况下，CAP应该会继续发挥作用。这些抑制剂可以作为一种佐剂用于 联合疗法和CAP治疗耐药感染。 在第一个资助期，我们确定了一组作为抗生素佐剂的化合物，增强了 对粘菌素敏感。在目前的工作中，我们建议表征这种组合的有效性 治疗(目标1)。我们将调查对生物膜、耐药菌株和耐药株的活性- 诱导条件。此外，还将研究杀灭动力学和抗药性的发展。我们目前的情况 假说是抗生素佐剂通过抑制Arna发挥作用，Arna是一种关键的双功能生物合成酶 脂类修饰途径。我们将研究佐剂对Arna介导的抗性的影响 通过质谱分析脂类和与Arna在动力学和动力学上的直接相互作用而实现的途径 结合实验(目标2)。拟议的工作将验证该组合的进一步发展 治疗走向临床使用。分子靶标的确定也将指导EVEN的优化 在未来的工作中会有更多有效的抑制剂。破译分子目标也可以让我们扩大 其他革兰氏阴性菌的治疗，如碳青霉烯类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糖尿病的治疗指数 粘菌素，最大限度地减少药物的严重副作用，以及使粘菌素耐药治疗成为可能 菌株，确保救命药物的持续疗效。

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.