Study of key RNA metabolism proteins in <i>Enterococcus faecalis</i>

The control of mRNA turnover is essential in bacteria to allow rapid adaptation, especially in opportunistic pathogen like <i>Enterococcus faecalis</i>. This mechanism involves RNase and DEAD-box helicases that are key elements in RNA processing and their associations form the degradosome with accessory proteins. In this study, we investigated the function of four RNases (J1, J2, Y and III) and three DEAD-box helicases (CshA, CshB, CshC) present in most Enterococci. The interactions of all these RNA metabolism actors were investigated <i>in vitro</i>, and the results are in accordance with a degradosome structure close to the one of <i>Bacillus subtilis</i>. At the physiological level, we showed that RNase J1 is essential, whereas RNases J2 and III have a role in cold, oxidative and bile salts stress response, and RNase Y in general fitness. Furthermore, RNases J2, Y and III mutants are affected in virulence in the <i>Galleria mellonella</i> infection model. Concerning DEAD-box helicases, all of them are involved in cold shock response. Since the Δ<i>cshA</i> mutant was the most stress impacted strain, we studied this DEAD-box helicase CshA in more detail. This showed that CshA autoregulates its own expression by binding to its mRNA 5ʹUnstranslated Region. Interestingly, CshC is also involved in the expression control of CshA by a hitherto unprecedented mechanism.信使RNA（mRNA）周转调控对于细菌实现快速适应至关重要，对于粪肠球菌（Enterococcus faecalis）这类机会致病菌而言尤为如此。该调控机制涉及核糖核酸酶（RNase）与DEAD-box解旋酶（DEAD-box helicases），二者均为RNA加工过程中的关键元件；这些元件与辅助蛋白组装形成降解体（degradosome）。本研究针对多数肠球菌属（Enterococcus）菌株中存在的4种核糖核酸酶（J1、J2、Y及III型）与3种DEAD-box解旋酶（CshA、CshB、CshC）的功能展开了探究。本研究对上述所有RNA代谢相关因子的相互作用开展了体外（in vitro）实验探究，结果显示其降解体结构与枯草芽孢杆菌（Bacillus subtilis）的降解体结构高度相似。在生理层面，本研究证实核糖核酸酶J1是必需的；而核糖核酸酶J2与III型则参与冷应激、氧化应激及胆汁盐应激响应，核糖核酸酶Y则与细菌的整体生存适应性相关。此外，核糖核酸酶J2、Y及III型的突变株在大蜡螟（Galleria mellonella）感染模型中的毒力表现受到显著影响。关于DEAD-box解旋酶，所有3种亚型均参与冷休克响应。由于ΔcshA突变株是受应激影响最为显著的菌株，本研究对该DEAD-box解旋酶CshA展开了更为深入的分析。研究结果显示，CshA可通过结合其mRNA的5'非翻译区（5'UTR）实现自身表达的自主调控。值得注意的是，CshC还通过一种迄今尚未见报道的机制参与CshA的表达调控。