Department of Biomolecular Health Sciences, Division Infectious Diseases & Immunology, Section Molecular Host Defence, Utrecht University, Utrecht, The Netherlands.
Department of Biomolecular Health Sciences, Division Infectious Diseases and Immunology, Section Immunology, Utrecht University, Utrecht, The Netherlands.
J Glob Antimicrob Resist. 2022 Sep;30:406-413. doi: 10.1016/j.jgar.2022.07.009. Epub 2022 Jul 12.
Our group recently developed a new group of antimicrobial peptides termed PepBiotics, of which peptides CR-163 and CR-172 showed optimized antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus without inducing antimicrobial resistance. In this study, the antibacterial mechanism of action and the immunomodulatory activity of these two PepBiotics was explored.
RAW264.7 cells were used to determine the ability of PepBiotics to neutralize Lipopolysaccharide (LPS)-and Lipoteichoic acid (LTA)-induced activation of macrophages. Isothermal titration calorimetry and competition assays with dansyl-labeled polymyxin B determined binding characteristics to LPS and LTA. Combined bacterial killing with subsequent macrophage activation assays was performed to determine so-called 'silent killing'. Finally, flow cytometry of peptide-treated genetically engineered Escherichia coli expressing Green Fluorescent Protein (GFP) and mCherry in the cytoplasm and periplasm, respectively, further established the antimicrobial mechanism of PepBiotics.
Both CR-163 and CR-172 were shown to have broad-spectrum activity against ESKAPE pathogens and E. coli using a membranolytic mechanism of action. PepBiotics could exothermically bind LPS/LTA and were able to replace polymyxin B. Finally, it was demonstrated that bacteria killed by PepBiotics were less prone to stimulate immune cells, contrary to gentamicin and heat-killed bacteria that still elicited a strong immune response.
These studies highlight the multifunctional nature of the two peptide antibiotics as both broad-spectrum antimicrobial and immunomodulator. Their ability to kill bacteria and reduce unwanted subsequent immune activation is a major advantage and highlights their potential for future therapeutic use.
我们小组最近开发了一组新的抗菌肽，称为 PepBiotics，其中肽 CR-163 和 CR-172 对铜绿假单胞菌和金黄色葡萄球菌表现出优化的抗菌活性，而不会诱导抗菌耐药性。在这项研究中，探索了这两种 PepBiotics 的抗菌作用机制和免疫调节活性。
使用 RAW264.7 细胞来确定 PepBiotics 中和脂多糖（LPS）和脂磷壁酸（LTA）诱导的巨噬细胞激活的能力。使用荧光标记的多粘菌素 B 进行等温滴定量热法和竞争测定来确定与 LPS 和 LTA 的结合特性。通过与随后的巨噬细胞激活测定相结合进行联合细菌杀伤，以确定所谓的“沉默杀伤”。最后，通过用肽处理分别在细胞质和周质中表达绿色荧光蛋白（GFP）和 mCherry 的基因工程大肠杆菌的流式细胞术，进一步确定了 PepBiotics 的抗菌机制。
CR-163 和 CR-172 均显示出针对 ESKAPE 病原体和大肠杆菌的广谱活性，其作用机制为膜溶解。PepBiotics 可以放热地结合 LPS/LTA 并能够替代多粘菌素 B。最后，证明被 PepBiotics 杀死的细菌不易刺激免疫细胞，与庆大霉素和热失活细菌形成对比，后者仍会引发强烈的免疫反应。
这些研究强调了这两种肽抗生素作为广谱抗菌和免疫调节剂的多功能性质。它们杀死细菌并减少不必要的后续免疫激活的能力是一个主要优势，突出了它们在未来治疗用途中的潜力。
