Microbial Biology, Ruhr University Bochum, Bochum, Germany.
Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany.
PLoS Pathog. 2021 Nov 12;17(11):e1009650. doi: 10.1371/journal.ppat.1009650. eCollection 2021 Nov.
Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5'-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.
许多细菌病原体使用 III 型分泌系统（T3SS）作为分子注射器将效应蛋白注入宿主细胞。在食源性病原体假结核耶尔森氏菌中，通过 T3SS 输送分泌的效应蛋白鸡尾酒依赖于 YopN，这是一种分子守门员，控制着从细菌细胞质进入分泌通道的通道。在这里，我们表明，有几个检查点根据毒力条件调整 yopN 的表达。一个主要的线索是宿主的体温。已知 37°C 的温度会诱导 RNA 温度计（RNAT）依赖性合成 LcrF，这是一种转录因子，可激活整个 T3SS 调节子的表达。在这里，我们发现了第二层温度控制。我们表明，另一个 RNAT 在环境温度较低时沉默 yopN mRNA 的翻译。两种细胞 yopN 同工型的长和短 5'-非翻译区折叠成相似的二级结构，阻止核糖体结合。发夹结构具有内部环，在 37°C 时熔化，从而如突变分析、体外结构探测和足迹分析方法所示，允许形成翻译起始复合物。重要的是，我们通过使用带有被捕获的 SD 序列的热稳定 RNAT 变体来证明该 RNAT 在 III 型分泌的忠实控制中的生理相关性。在该菌株中，YopN 产量的减少导致效应蛋白不受限制地分泌到培养基中，细菌生长停滞，并延迟进入真核宿主细胞的易位。总的来说，我们的结果表明，Yersinia T3SS 的底物输送受到两个 RNAT 的分层监测。
