Antimicrobial resistance is undoubtedly one of the greatest global health threats. The emergence of multidrug-resistant (MDR) Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactamase-resistant Streptococcus pneumonia, has severely limited our antibiotic arsenal. Numerous ribosome-targeting antibiotics, especially pleuromutilins, oxazolidinones, and streptogramins, are viewed as promising alternatives against aggressive MDR pathogens. In this study, we identified a new adenosine triphosphate (ATP)-binding cassete (ABC)-F family determinant, srpA, in Streptococcus suis (S. suis) by means of a comparative analysis of the whole-genome sequences of tiamulin (TIA)-resistant and TIA-sensitive bacteria. Functional cloning confirmed that the deduced gene can mediate cross-resistance to pleuromutilins, lincosamides, and streptogramin A in S. suis and S. aureus. A sequence alignment revealed that SrpA shares the highest amino acid identity with Vga(E) (36%) and shows canonical characteristics of ABC-F family members. In SrpA-ribosome docked compounds, the extended loop region of SrpA approaches the valnemulin-binding pocket in the ribosome peptidyl-transferase center and competes with bound valnemulin. A detailed mutational analysis of the loop residues confirmed that this domain is crucial for SrpA activity, as substitutions or truncations of this region affect the efficiency and specificity of antibiotic resistance. Intracellular antibiotics accumulation indicated that SrpA does not act as an efflux pump, while a ribosome binding assay supported the protective effects of SrpA on the ribosome by preventing antibiotic binding as well as displacing bound drugs. These findings clarify the mechanisms underlying resistance to ribosomal antibiotics.

抗菌素耐药性无疑是全球最大的健康威胁之一。耐多药 (MDR) 革兰氏阳性病原体的出现，例如耐甲氧西林金黄色葡萄球菌(MRSA)、耐万古霉素屎肠球菌(VRE) 和耐β-内酰胺酶肺炎链球菌，严重限制了我们的抗生素库。许多靶向核糖体的抗生素，特别是截短侧耳素、恶唑烷酮和链霉素，被视为对抗侵袭性 MDR 病原体的有希望的替代品。在这项研究中，我们在猪链球菌( S. suis) 通过对 tiamulin (TIA) 抗性和 TIA 敏感细菌的全基因组序列进行比较分析。功能性克隆证实，推断的基因可以介导S. suis和S. aureus对截短侧耳素、林可酰胺和链霉素 A 的交叉抗性. 序列比对显示 SrpA 与 Vga(E) (36%) 具有最高的氨基酸同一性，并显示出 ABC-F 家族成员的典型特征。在 SrpA-核糖体对接的化合物中，SrpA 的扩展环区接近核糖体肽基转移酶中心的 valnemulin 结合口袋并与结合的 valnemulin 竞争。对环残基的详细突变分析证实，该结构域对 SrpA 活性至关重要，因为该区域的替换或截断会影响抗生素抗性的效率和特异性。细胞内抗生素积累表明 SrpA 不充当外排泵，而核糖体结合试验通过阻止抗生素结合以及取代结合药物来支持 SrpA 对核糖体的保护作用。