Characterization of novel small RNAs (sRNAs) contributing to the desiccation response of Salmonella enterica serovar Typhimurium

Noncoding RNA (ncRNA) modulation of gene expression has now been ubiquitously observed across all domains of life. An increasingly apparent role of ncRNAs is to coordinate changes in gene expressions in response to environmental stress. Salmonella enterica, a common food-born pathogen, is known for its striking ability to survive, adapt, and thrive in various unfavourable environments which makes it a particularly difficult pathogen to eliminate as well as an interesting model in which to study ncRNA contributions to cellular stress response. Mounting evidence now suggests that small RNAs (sRNAs) represent key regulators of Salmonella stress adaptation. Approximately 50–500 nucleotides in length, sRNAs regulate gene expression through complementary base pairing with molecular targets and have recently been suggested to outnumber protein-coding genes in bacteria. In this work, we employ small RNA transcriptome sequencing to characterize changes in the sRNA profiles of Salmonella in response to desiccation. In all, we identify 102 previously annotated sRNAs significantly differentially expressed during desiccation; and excitingly, 71 novel sRNAs likewise differentially expressed. Small transcript northern blotting and qRT-PCRs confirm the identities and expressions of several of our novel sRNAs, and computational analyses indicate the majority are highly conserved and structurally related to characterized sRNAs. Predicted sRNA targets include several proteins necessary for desiccation survival and this, in part, suggests a role for desiccation-regulated sRNAs in this stress response. Furthermore, we find individual knock-outs of two of the novel sRNAs identified herein, either sRNA1320429 or sRNA3981754, significantly impairs the ability of Salmonella to survive desiccation, confirming their involvements (and suggesting the potential involvements of other sRNAs we identify in this work) in the Salmonella response to desiccation.

非编码RNA（Noncoding RNA, ncRNA）对基因表达的调控作用，如今已在所有生命域中被广泛观测到。非编码RNA日益明确的重要功能之一，是协调基因表达变化以响应环境胁迫。肠炎沙门氏菌（Salmonella enterica）是一种常见食源性致病菌，因其能在多种不利环境中存活、适应并增殖的卓越能力而闻名——这不仅使其成为极难清除的致病菌，也为研究非编码RNA在细胞胁迫应答中的作用提供了极具价值的模型。越来越多的证据表明，小型RNA（small RNAs, sRNAs）是沙门氏菌胁迫适应的关键调控因子。小型RNA长度约为50至500个核苷酸，通过与分子靶点互补碱基配对调控基因表达；近年来的研究显示，细菌中的小型RNA数量已超过编码蛋白质的基因。本研究采用小型RNA转录组测序技术，对沙门氏菌在脱水胁迫下的sRNA表达谱变化进行表征。总计，我们鉴定出102个此前已注释的sRNAs在脱水胁迫过程中显著差异表达；令人振奋的是，另有71个全新sRNAs同样呈现出差异表达特征。小分子RNA Northern印迹（Northern blotting）与实时定量逆转录PCR（qRT-PCR）实验验证了部分全新sRNAs的序列与表达情况；生物信息学分析显示，其中大多数序列具有高度保守性，且结构与已被表征的sRNAs高度相似。预测得到的sRNA靶点包含多种与脱水存活相关的必需蛋白质，这在一定程度上表明，受脱水调控的sRNAs参与了该胁迫应答过程。此外，我们对本研究鉴定出的两个全新sRNAs（sRNA1320429与sRNA3981754）分别进行敲除实验，结果显示两者的敲除均会显著削弱沙门氏菌的脱水存活能力，这证实了这两个sRNAs的参与作用，并提示本研究中鉴定的其他sRNAs也可能参与沙门氏菌的脱水胁迫应答。