d'Acoz Ophélie d'Udekem, Hue Fong, Ye Tianyi, Wang Louise, Leroux Maxime, Rajngewerc Lucila, Tran Tung, Phan Kimberly, Ramirez Maria S, Reisner Walter, Tolmasky Marcelo E, Reyes-Lamothe Rodrigo
Department of Biology, McGill University, 3649 Sir William Osler, Montréal, Québec, H3G 0B1, Canada.
Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, California.
bioRxiv. 2023 Dec 15:2023.09.05.556435. doi: 10.1101/2023.09.05.556435.
Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in , and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.
氨基糖苷类药物是现有治疗细菌感染药物库中的重要组成部分。耐药基因的激增和快速传播极大地降低了它们的疗效，对公众健康构成威胁。在众多赋予对氨基糖苷类药物耐药性的修饰酶中，氨基糖苷类乙酰转移酶AAC(6')-Ib最为普遍且在临床环境中最为相关，因为它能使多种氨基糖苷类药物失活，如阿米卡星。尽管对AAC(6')-Ib蛋白的作用机制、结构和生化特性已进行了广泛研究，但其细胞内环境对其活性的影响仍不清楚。在这项研究中，我们使用基于荧光的系统来量化细胞内每个细胞中AAC(6')-Ib的数量，并通过CRISPR干扰方法调节这个拷贝数。然后利用这些工具将酶浓度与对阿米卡星的耐药水平相关联。我们的结果表明，对阿米卡星的耐药性随着AAC(6')-Ib浓度的升高呈线性增加，直到在特定蛋白质浓度下达到平台期。对该蛋白的成像显示，它在细胞胞质内自由扩散，但在丰富培养基中浓度较高时倾向于形成包涵体。添加螯合剂可完全溶解这些聚集体，并部分阻止耐药水平的平台期，这表明AAC(6')-Ib聚集体会降低对阿米卡星的耐药性。这些结果为理解每个AAC(6')-Ib分子对氨基糖苷类耐药性的细胞影响提供了第一步。它们还突出了研究其在细胞内动态行为的重要性。
