Oriol Charlotte, Cengher Liviu, Manna Adhar C, Mauro Tony, Pinel-Marie Marie-Laure, Felden Brice, Cheung Ambrose, Rouillon Astrid
Rennes 1 University, INSERM, BRM [Bacterial Regulatory RNAs and Medicine], UMR_S 1230, Rennes, France.
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
mSystems. 2021 Oct 26;6(5):e0071321. doi: 10.1128/mSystems.00713-21. Epub 2021 Oct 12.
SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a Δ mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of , a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and methodologies are effective to characterize transcriptional factor (TF) regulons and functions. Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of , a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.
SarA是金黄色葡萄球菌的一种转录调节因子，是一个主要的全局调节系统，可协调参与其致病性的靶基因的表达。各种研究已经鉴定出大量的SarA靶基因，但尚未对包括小调节RNA（sRNA）在内的调控子进行深入表征。在本研究中，我们利用转录组测序（RNA-Seq）和染色质免疫沉淀测序（ChIP-Seq）来确定SarA调控靶标的完整列表，包括mRNA和sRNA。RNA-Seq分析表明，在Δ突变体中有390个mRNA和51个sRNA差异表达，而ChIP-Seq揭示了金黄色葡萄球菌基因组中的354个mRNA和55个sRNA靶标。我们通过Northern印迹和电泳迁移率变动分析证实了几个新的SarA靶标的真实性。其中，我们对编码I型毒素-抗毒素系统毒素的基因的抑制作用进行了表征，表明SarA及其同源抗毒素SprF2对毒素表达进行了多层锁定。最后，利用15个新的SarA调控的sRNA靶标的上游启动子序列生成了一个新的SarA共有DNA结合序列。用推导的SarA基序进行全基因组扫描，发现了在我们的RNA-Seq和ChIP-Seq分析中未鉴定的新的潜在SarA靶基因。用一个预测的sRNA靶标证实了这个新共有序列的强度。RNA-Seq和ChIP-Seq组合分析给出了调控的快照，而生物信息学分析则基于序列揭示了靶标的永久视图。总之，这些实验和方法对于表征转录因子（TF）调控子和功能是有效的。金黄色葡萄球菌是一种共生兼机会性病原体，可导致大量从良性到严重的人类和动物感染。感染期间的基因表达适应需要一个复杂的调节网络，包括转录因子（TF）和sRNA。TF SarA影响毒力、代谢、生物膜形成和对某些抗生素的抗性。SarA直接调节约20个mRNA和一些sRNA的表达。在这里，我们将高通量表达筛选方法与结合测定和生物信息学相结合，对SarA调控子进行了深入研究。这种组合方法鉴定出了85个前所未有的mRNA和sRNA靶标，其中至少14个是主要靶标。在新的SarA直接靶标中，我们对编码毒素-抗毒素系统毒素的基因的抑制作用进行了表征，表明SarA及其同源抗毒素SprF2对毒素表达进行了多层锁定。
