Močibob Marko, Obranić Sonja, Kifer Domagoj, Rokov-Plavec Jasmina, Maravić-Vlahoviček Gordana
University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Biochemistry and Molecular Biology, A. Kovačića 1, 10000, Zagreb, Croatia; University of Zagreb, Faculty of Science, Department of Chemistry, Horvatovac 102a, 10000, Zagreb, Croatia.
University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Biochemistry and Molecular Biology, A. Kovačića 1, 10000, Zagreb, Croatia; University North, University Centre Varaždin, 104. brigade 1, 42000, Varaždin, Croatia.
Arch Biochem Biophys. 2025 Jul;769:110422. doi: 10.1016/j.abb.2025.110422. Epub 2025 Apr 11.
Aminoglycosides are broad-spectrum antibiotics critical to clinical treatment, but the emergence of bacterial resistance, particularly through 16S rRNA methyltransferases, has compromised their efficacy. These enzymes, originally discovered in natural aminoglycoside producers, confer resistance by methylating nucleotides G1405 and A1408 in 16S rRNA, blocking antibiotic binding to the ribosome. This study investigated the binding affinities and methylation activities of 16S rRNA methyltransferases KamB, NpmA, RmtA, RmtC, and Sgm with immature 30S ribosomal subunits from E. coli strains lacking RimM and YjeQ ribosomal assembly factors. Binding affinities to mature 30S ribosomal subunits and immature 30S assembly forms isolated from ΔyjeQ and ΔrimM strains were determined by microscale thermophoresis and interactions were further validated with in vitro pull-down assays. Methylation of immature 30S subunits was examined with primer extension on 16S rRNA extracted from methylation assays in vitro and from cells with immature 30S subunits expressing 16S rRNA methyltransferases in vivo, showing successful methylation of target nucleotides in both experimental systems. The results reveal that aminoglycoside resistance methyltransferases are capable to bind and modify late-stage immature 30S ribosomal subunits pointing to possibility that the resistance to aminoglycoside antibiotics is installed and established before the full maturation of ribosomal 30S subunit.
氨基糖苷类抗生素是临床治疗中至关重要的广谱抗生素，但细菌耐药性的出现，尤其是通过16S rRNA甲基转移酶产生的耐药性，已经损害了它们的疗效。这些酶最初是在天然氨基糖苷类抗生素产生菌中发现的，通过甲基化16S rRNA中的核苷酸G1405和A1408赋予耐药性，从而阻止抗生素与核糖体结合。本研究调查了16S rRNA甲基转移酶KamB、NpmA、RmtA、RmtC和Sgm与缺乏RimM和YjeQ核糖体组装因子的大肠杆菌菌株的未成熟30S核糖体亚基的结合亲和力和甲基化活性。通过微量热泳测定了与从ΔyjeQ和ΔrimM菌株中分离的成熟30S核糖体亚基和未成熟30S组装形式的结合亲和力，并通过体外下拉试验进一步验证了相互作用。通过对体外甲基化试验以及体内表达16S rRNA甲基转移酶的具有未成熟30S亚基的细胞中提取的16S rRNA进行引物延伸，检测未成熟30S亚基的甲基化，结果表明在两个实验系统中靶核苷酸均成功甲基化。结果显示，氨基糖苷类耐药甲基转移酶能够结合并修饰后期未成熟的30S核糖体亚基，这表明对氨基糖苷类抗生素的耐药性可能在核糖体30S亚基完全成熟之前就已产生并确立。
