"Deep eutectic solvent enhances antibacterial activity of a modular lytic enzyme against Acinetobacter baumannii " by Aleksandra Maria Kocot, Tomasz Swebocki, Karolina Ciemińska, Adrianna Łupkowska, Małgorzata Kapusta, Dennis Grimon, Ewa Laskowska, Anna-Karina Kaczorowska, Tadeusz Kaczorowski, Rabah Boukherroub, Yves Briers, Magdalena Plotka

In this study, we evaluated the combined effect between MLE-15, a modular lytic enzyme composed of four building blocks, and reline, a natural deep eutectic solvent. The bioinformatic analysis allowed us to determine the spatial architecture of MLE-15, whose components were bactericidal peptide cecropin A connected via a flexible linker to the cell wall binding domain (CBD) of mesophilic 201ϕ2-1 endolysin and catalytic domain EAD of highly thermostable Ph2119 endolysin. The modular enzyme showed high thermostability with the melting temperature of 93.97 ± 0.38°C, significantly higher than their natural counterparts derived from mesophilic sources. The minimum inhibitory concentration (MIC) of MLE-15 was 100 µg/mL for a panel of Gram-positive and Gram-negative bacteria, while the MIC of reline ranged from 6.25% to 25% v/v for the same strains. The addition of reline effectively reduced the MIC of MLE-15 from 3.15 to 50 µg/mL. This combination displayed additive effects for most strains and synergism for extensively antibiotic-resistant Acinetobacter baumannii and Bacillus subtilis. The subsequent evaluation revealed that MLE-15 eliminated planktonic cells of A. baumannii RUH134, but was ineffective against matured biofilms. However, combined with reline, MLE-15 reduced the bacterial load in the matured biofilm by 1.39 log units. Confocal fluorescence microscopy indicated that reline damaged the structure of the biofilm, allowing MLE-15 to penetrate it. Additionally, MLE-15 and its combination with reline entirely prevented the formation of a biofilm and eradicated meropenem-persistent cells of A. baumannii RUH134. Effectiveness in lowering the MIC value of MLE-15 as well as complete protection against biofilm formation and antibiotic-tolerant persister cells, indicate that MLE-15 and reline combination is a promising candidate for effective therapies in bacterial infections, which is especially important in the light of the global crisis of antimicrobial resistance.

本研究评估了由四个结构单元构成的模块化裂解酶MLE-15与天然低共熔溶剂reline的联合效应。通过生物信息学分析，我们明确了MLE-15的空间结构：其组分包括杀菌肽天蚕素A（cecropin A），该肽通过柔性连接子与嗜温201ϕ2-1内溶素的细胞壁结合结构域（cell wall binding domain, CBD）以及高耐热性Ph2119内溶素的催化结构域EAD相连。该模块化裂解酶展现出优异的热稳定性，其解链温度为93.97±0.38℃，显著高于来源于嗜温菌株的天然同源酶。针对一组革兰氏阳性菌与革兰氏阴性菌，MLE-15的最低抑菌浓度（minimum inhibitory concentration, MIC）为100 μg/mL；而针对同一批菌株，reline的最低抑菌浓度范围为6.25%~25%（体积比, v/v）。加入reline可有效将MLE-15的最低抑菌浓度从3.15 μg/mL降至50 μg/mL。该联合方案对大多数菌株表现出相加效应，而对泛抗生素耐药鲍曼不动杆菌（Acinetobacter baumannii）与枯草芽孢杆菌（Bacillus subtilis）则展现出协同效应。后续评估结果显示，MLE-15可清除鲍曼不动杆菌RUH134的浮游菌，但无法作用于成熟生物被膜。但当与reline联用时，MLE-15可将成熟生物被膜内的细菌载量降低1.39个对数单位。共聚焦荧光显微镜观测结果表明，reline可破坏生物被膜结构，使MLE-15得以穿透被膜。此外，MLE-15单独使用及其与reline联合使用时，均可完全抑制鲍曼不动杆菌RUH134生物被膜的形成，并根除其美罗培南耐药持留菌（meropenem-persistent cells）。该联合方案既能有效降低MLE-15的最低抑菌浓度，又可完全抑制生物被膜形成并清除抗生素耐受持留菌，表明MLE-15与reline的联合方案是细菌性感染有效治疗的极具潜力的候选方案，在全球抗菌药物耐药性危机的背景下，该方案的开发尤为重要。