The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5′-UTR Variant

Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5′-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5′-untranslated regions (5′-UTR) of 28 and 234 nucleotides. The transcript containing the long 5′-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5′-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5′-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5′-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5′-UTR. These findings emphasize how 5′-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5′-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.单核细胞增生李斯特菌（Listeria monocytogenes）是一种细菌性致病菌，其基因组编码多种可与肽聚糖（peptidoglycan）共价结合的细胞壁蛋白。该蛋白家族的部分成员在致病过程中发挥关键作用，近期研究表明，其中部分蛋白（如Lmo0514）在定殖真核细胞的细菌中表达上调。目前，介导这些细胞壁蛋白表达变化的调控机制仍未得到充分阐释。本研究针对胞内细菌中Lmo0514蛋白水平升高的调控机制展开探究。研究发现，该蛋白在胞内细菌中的含量显著升高（增幅超200倍），远高于lmo0514转录本的表达增幅（约6倍）。通过5'-cDNA末端快速扩增（Rapid Amplification of 5′-cDNA Ends, RACE）实验，我们鉴定出两种lmo0514转录本，其5'非翻译区（5′-untranslated regions, 5′-UTR）长度分别为28和234个核苷酸。其中携带较长5'UTR的转录本可在胞内细菌中被上调。234个核苷酸的长5'UTR同时也是小RNA（small RNA, sRNA）Rli27的作用靶点——我们通过生物信息学分析发现，该sRNA与长5'UTR存在广泛的碱基配对潜力。预测显示，这种相互作用可解除长5'UTR对夏因-达尔加诺序列（Shine-Dalgarno sequence）的遮蔽，从而促进真核细胞内Lmo0514蛋白的合成。实时定量聚合酶链式反应（Real-time quantitative PCR）结果表明，Rli27在胞内细菌中表达上调。体内实验显示，缺失Rli27的胞内细菌中Lmo0514蛋白水平显著降低；通过表达野生型Rli27分子可恢复Lmo0514的野生型表达水平，但无法与lmo0514长5'UTR结合的突变型Rli27则无此效果。本研究结果揭示了5'UTR长度如何通过特定sRNA调控基因表达，同时阐明了两种携带不同5'UTR的转录本的相对丰度变化，如何将靶基因sRNA的调控作用限定于占据真核细胞内微环境的细菌中。