Coupling tRNAGly gene redundancy with staphylococcal cell wall integrity, antibiotic susceptibility and virulence potential

Redundancy in the number of tRNA genes which occur across species is poorly understood and, in many cases, remains essentially unexplored. In Staphylococcus aureus, several tRNAGly genes encode isoacceptors that either participate in protein synthesis or cell wall formation, upon aminoacylation by the sole glycyl-tRNA synthetase (GlyRS). Transcription of GlyRS, is regulated by a glyS T-box riboswitch in the 5'UTR, which responds to all tRNAGly isoacceptors and exhibits species-specific RNA conformations. To address whether disruption of the tRNAGly expression impacts the balance between ribosomal translation and cell wall formation, we used CRISPR/Cas9 editing to ablate one proteinogenic tRNAGly gene copy which is the strongest ligand of the glyS T-box riboswitch. Interestingly, no discernible impact on the growth and general translational activity of S. aureus was observed, suggesting that the remaining tRNAs could make up for this deficiency. However, transcriptomics and proteomics analyses revealed that the edited strain had deficient cell wall integrity, and subsequent experimentation showed increased susceptibility to antibiotics targeting the cell wall and resistance to lysostaphin. Interestingly, the observed alteration in the proteome level were independent of the glycine codon usage. Moreover, the edited strain exhibited reduced biofilm formation but retained its ability to invade human cell cultures. Overall, the present study highlights the important role of tRNA gene copy redundancy in the physiology of an important pathogen like S. aureus and consolidates the regulatory role of tRNAs in metabolic homeostasis. Overall design: Expression profiling by the Ion Torrent Genestudio S5 platform with Ion Chef (Ion torrent) and Illumina NextSeq 1000 platform.

跨物种存在的tRNA(transfer RNA)基因数量冗余现象的相关机制尚不明确，且在多数情况下几乎未得到深入探索。在金黄色葡萄球菌(Staphylococcus aureus)中，多个甘氨酸tRNA(tRNAGly)基因编码的同工受体可在仅由甘氨酰-tRNA合成酶(GlyRS)完成氨酰化修饰后，分别参与蛋白质合成与细胞壁形成过程。甘氨酰-tRNA合成酶(GlyRS)的转录受5'非翻译区(5'UTR)内的glyS T盒核糖开关(glyS T-box riboswitch)调控，该开关可识别所有甘氨酸tRNA同工受体，并呈现物种特异性的RNA构象。为探究甘氨酸tRNA表达紊乱是否会影响核糖体翻译与细胞壁合成之间的平衡，我们采用CRISPR/Cas9基因编辑技术，敲除了一个作为glyS T盒核糖开关最强配体的、可编码蛋白质的甘氨酸tRNA基因拷贝。令人意外的是，未观察到该操作对金黄色葡萄球菌的生长及整体翻译活性产生可检测的影响，这表明剩余的tRNA可弥补该基因缺失带来的缺陷。但转录组学与蛋白质组学分析显示，编辑后的菌株细胞壁完整性存在缺陷；后续实验还发现，该菌株对靶向细胞壁的抗生素易感性升高，同时对溶葡萄球菌素产生抗性。值得注意的是，所观测到的蛋白质组水平变化与甘氨酸密码子使用偏好无关。此外，编辑后的菌株生物被膜形成能力下降，但仍保留侵袭人类细胞培养物的能力。综上，本研究阐明了tRNA基因拷贝数冗余在金黄色葡萄球菌这类重要病原菌生理过程中的关键作用，并证实了tRNA在代谢稳态中的调控功能。实验设计概述：采用搭载Ion Chef的Ion Torrent Genestudio S5平台（Ion torrent）与Illumina NextSeq 1000平台开展表达谱分析。