Data_Sheet_5_Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Staphylococcus aureus Under Daptomycin Exposure.PDF

Daptomycin (DAP) is one of the last-resort treatments for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) infections. DAP resistance (DAP-R) is multifactorial and mainly related to cell-envelope modifications caused by single-nucleotide polymorphisms and/or modulation mechanisms of transcription emerging as result of a self-defense process in response to DAP exposure. Nevertheless, the role of these adaptations remains unclear. We aim to investigate the comparative genomics and late post-exponential growth-phase transcriptomics of two DAP-resistant/DAP-susceptible (DAPR/S) methicillin-resistant S. aureus (MRSA) clinical strain pairs to focalize the genomic and long-term transcriptomic fingerprinting and adaptations related to the DAP mechanism of action acquired in vivo under DAP pressure using Illumina whole-genome sequencing (WGS), RNA-seq, bioinformatics, and real-time qPCR validation. Comparative genomics revealed that membrane protein and transcriptional regulator coding genes emerged as shared functional coding-gene clusters harboring mutational events related to the DAP-R onset in a strain-dependent manner. Pairwise transcriptomic enrichment analysis highlighted common and strain pair-dependent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, whereas DAPR/S double-pair cross-filtering returned 53 differentially expressed genes (DEGs). A multifactorial long-term transcriptomic-network characterized DAPR MRSA includes alterations in (i) peptidoglycan biosynthesis, cell division, and cell-membrane (CM) organization genes, as well as a cidB/lytS autolysin genes; (ii) ldh2 involved in fermentative metabolism; (iii) CM-potential perturbation genes; and (iv) oxidative and heat/cold stress response-related genes. Moreover, a D-alanyl–D-alanine decrease in cell-wall muropeptide characterized DAP/glycopeptide cross-reduced susceptibility mechanisms in DAPR MRSA. Our data provide a snapshot of DAPR MRSA genomic and long-term transcriptome signatures related to the DAP mechanism of action (MOA) evidencing that a complex network of genomic changes and transcriptomic adaptations is required to acquire DAP-R.

达托霉素（Daptomycin, DAP）是治疗异质性万古霉素中介金黄色葡萄球菌（heterogeneous vancomycin-intermediate Staphylococcus aureus, hVISA）和万古霉素中介金黄色葡萄球菌（vancomycin-intermediate S. aureus, VISA）感染的最后一线疗法之一。达托霉素耐药（DAP resistance, DAP-R）具有多因素性，主要与单核苷酸多态性（single-nucleotide polymorphisms）引发的细胞包膜修饰，以及因暴露于达托霉素的自我防御过程中产生的转录调控机制相关。不过，这些适应性改变的作用仍不明确。本研究旨在通过Illumina全基因组测序（whole-genome sequencing, WGS）、RNA测序（RNA-seq）、生物信息学分析及实时定量PCR（real-time qPCR）验证，对两株耐/敏达托霉素（DAP-resistant/DAP-susceptible, DAPR/S）耐甲氧西林金黄色葡萄球菌（methicillin-resistant S. aureus, MRSA）临床菌株对的比较基因组学和晚期指数生长后阶段转录组学进行研究，以聚焦体内在达托霉素压力下获得的、与达托霉素作用机制相关的基因组及长期转录组特征谱与适应性改变。比较基因组学分析显示，编码膜蛋白与转录调控因子的基因作为共享的功能编码基因簇，以菌株依赖的方式携带与DAP-R发生相关的突变事件。成对转录组富集分析突出了共同的及菌株对依赖的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路，而DAPR/S双菌株对的交叉筛选得到了53个差异表达基因（differentially expressed genes, DEGs）。一个多因素的长期转录组网络表征了DAPR MRSA的特征，其包含：（i）肽聚糖生物合成、细胞分裂及细胞膜（cell-membrane, CM）组织相关基因，以及cidB/lytS自溶素基因；（ii）参与发酵代谢的ldh2基因；（iii）潜在细胞膜扰动相关基因；（iv）氧化应激及冷热应激应答相关基因。此外，细胞壁肽聚糖中D-丙氨酰-D-丙氨酸含量的降低，构成了DAPR MRSA中达托霉素/糖肽类交叉敏感性降低的机制。本研究数据为与达托霉素作用机制（mechanism of action, MOA）相关的DAPR MRSA基因组及长期转录组特征提供了快照，证明获得DAP-R需要复杂的基因组改变与转录组适应性改变构成的调控网络。