Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu, 233030, China.
Department of Clinical Laboratory, Shanghai Clinical Medical College of Qingdao University, Shanghai Deji Hospital, Shanghai, 200331, China.
Microb Pathog. 2023 Oct;183:106325. doi: 10.1016/j.micpath.2023.106325. Epub 2023 Aug 26.
Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a leading public health problem, and is increasingly being reported worldwide with resistance to a wide spectrum of antibiotics. Recent reports have demonstrated that the outer membrane vesicles (OMVs) of gram-negative bacteria are potent resistance factors, but their role in the drug resistance of CRKP has not been elucidated. In order to investigate the effects of OMV components on drug resistance and to explore the mechanism of antimicrobial resistance in CRKP, we isolated the OMVs through ultracentrifugation, separated the OMV proteins through mass spectrometry (MS), and performed bioinformatics analysis. A total of 3,192 proteins were detected by nano LC-MS/MS analysis, with 108 (61.4%) cytoplasmic proteins, 50 (28.4%) cytoplasmic membrane proteins, nine (5.1%) periplasmic proteins, six (3.4%) outer membrane proteins, two (1.1%) extracellular proteins, and one (0.6%) other protein detected in the vesicles. MdtQ was detected as the only multidrug resistance outer membrane protein. Further experiments confirmed that MdtQ included the 1440 BP sequence and had a unique three-dimensional structure. To superimpose MdtQ with KPC-2 resistant proteins, I7ACB1, I7AKP2, and Q93LQ9, the root mean square deviation (RMSD) values were calculated (0.379, 0.671, and 1.35, respectively). I7ACB1 had the lowest RMSD value, indicating that it had the best superimposition effect. Furthermore, MdtQ had 20 biological pocket structures, and the four most important pockets were evenly distributed around the inner perimeter of its three-dimensional structure. These findings may provide a theoretical basis for controlling the spread of bacterial resistance in the future.
耐碳青霉烯类肺炎克雷伯菌（CRKP）已成为一个主要的公共卫生问题，在世界范围内，其对广谱抗生素的耐药性日益受到关注。最近的报告表明，革兰氏阴性菌的外膜囊泡（OMVs）是强有力的耐药因素，但它们在 CRKP 耐药性中的作用尚未阐明。为了研究 OMV 成分对耐药性的影响，并探讨 CRKP 中抗菌药物耐药的机制，我们通过超速离心法分离 OMVs，通过质谱（MS）分离 OMV 蛋白，并进行生物信息学分析。通过纳升 LC-MS/MS 分析共检测到 3192 种蛋白质，其中 108 种（61.4%）为细胞质蛋白，50 种（28.4%）为细胞质膜蛋白，9 种（5.1%）为周质蛋白，6 种（3.4%）为外膜蛋白，2 种（1.1%）为细胞外蛋白，1 种（0.6%）为囊泡中的其他蛋白。检测到 MdtQ 是唯一的多药耐药外膜蛋白。进一步的实验证实，MdtQ 包含 1440BP 序列，具有独特的三维结构。为了将 MdtQ 与 KPC-2 耐药蛋白 I7ACB1、I7AKP2 和 Q93LQ9 进行叠加，计算了均方根偏差（RMSD）值（分别为 0.379、0.671 和 1.35）。I7ACB1 的 RMSD 值最低，表明其叠加效果最好。此外，MdtQ 有 20 个生物口袋结构，其中最重要的四个口袋均匀分布在其三维结构的内周界周围。这些发现可能为未来控制细菌耐药性的传播提供理论依据。
