University of Würzburg, Institute of Molecular Infection Biology, Würzburg, Germany.
PLoS Pathog. 2019 Mar 14;15(3):e1007618. doi: 10.1371/journal.ppat.1007618. eCollection 2019 Mar.
RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-programming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities.
RsaE 是一种保守的小调控 RNA（sRNA），先前的研究表明它是金黄色葡萄球菌和枯草芽孢杆菌中碳代谢和其他代谢途径的核糖开关。在这里，我们发现 RsaE 有助于表皮葡萄球菌高变株中外源 DNA（eDNA）的释放和生物膜基质向多糖胞间黏附素（PIA）产生的转变。通过差异 RNA 测序（dRNA-seq）结合共聚焦激光扫描显微镜（CLSM）和报告基因融合的转录组分析表明，表皮葡萄球菌蛋白和 PIA 生物膜基质产生者在 RsaE 和代谢基因表达方面存在差异。RsaE 在表皮葡萄球菌 PIA 介导的生物膜中具有时空表达性，其过表达会触发 PIA 生物膜表型以及在表皮葡萄球菌蛋白生物膜基质产生株背景下 eDNA 的释放。dRNA-seq 和 Northern blot 分析表明，RsaE 主要以全长 100nt 的转录本和大约 5'-端缺少 20 个核苷酸的加工形式存在。RsaE 的加工导致 mRNA 靶标谱的扩展。因此，全长 RsaE 与表皮葡萄球菌抗孔蛋白编码 lrgA mRNA 相互作用，促进细菌裂解和 eDNA 释放。然而，加工后的 RsaE 与 icaR 和 sucCD mRNA 的 5'-UTR 相互作用，分别编码生物膜操纵子的阻遏物 IcaR 和三羧酸（TCA）循环中的琥珀酰辅酶 A 合成酶。RsaE 增强了 PIA 介导的生物膜基质产生，很可能是通过抑制 icaR 来激活 icaADBC 操纵子表达，以及通过抑制 TCA 循环和重新编程金黄色葡萄球菌的中心碳代谢来合成 PIA 前体。此外，RsaE 通过介导 eDNA 的释放作为稳定生物膜基质成分来支持生物膜的形成。由于 RsaE 本身在生物膜中不均匀表达，我们认为这种 sRNA 作为一种有利于表型异质性的因素，并支持表皮葡萄球菌生物膜群落中的分工。
