Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.

中文翻译：

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RNA温度计指示分泌细菌毒素的产生。

细菌病原体在其温血宿主和外部贮库之间的频繁转移伴随着温度的突然变化。37°C的温度可作为哺乳动物宿主摄入的可靠信号，从而引起细菌基因表达和代谢的重大重编程。肠胃耶尔森氏菌是革兰氏阴性病原体，可引起自限性胃肠道感染。在伪结核耶尔森氏菌的温度调节毒力基因中，cnfY编码细胞毒性坏死因子（CNFY），这是一种多功能的分泌毒素，可调节宿主的先天免疫系统，并有助于在急性感染和持久性之间做出决定。我们报告，温度调节cnfY表达的主要决定因素是5'端的热不稳定RNA结构。-非翻译区（5'-UTR）。各种翻译基因融合表明，该区域忠实地调节翻译起始，而与转录起始位点，启动子或报道基因株无关。RNA结构探测显示了不稳定的茎环结构，其中核糖体结合位点在25°C时被部分阻塞，但在37°C时释放。与细菌中的翻译控制一致，体外脚印（引物延伸抑制）实验表明，在高温下核糖体结合增加。将5'-UTR锁定在其25°C结构中的点突变会削弱茎环的打开，核糖体的进入和37°C的翻译起始。为了评估体内温度控制的相关性，我们使用了小鼠感染模型。是的 在cnfY上游携带稳定的RNA温度计变异体的假结核菌菌株无毒并且传播至肠系膜淋巴结和脾脏的能力减弱。我们以一个模型作为结论，在该模型中，RNA温度计在两层调节级联反应中充当翻译障碍，在急性感染期间严格控制CNFY毒素的提供。各种cnfY同源物上游的相似RNA结构表明，RNA热传感器决定了多种病原体中分泌毒素的产生。