Dual role of CsrA in regulating the hemolytic activity of <i>Escherichia coli</i> O157:H7

Post-transcriptional global carbon storage regulator A (CsrA) is a sequence-specific RNA-binding protein involved in the regulation of multiple bacterial processes. Hemolysin is an important virulence factor in the enterohemorrhagic <i>Escherichia coli</i> O157:H7 (EHEC). Here, we show that CsrA plays a dual role in the regulation of hemolysis in EHEC. CsrA significantly represses plasmid-borne enterohemolysin (EhxA)-mediated hemolysis and activates chromosome-borne hemolysin E (HlyE)-mediated hemolysis through different mechanisms. RNA structure prediction revealed a well-matched stem-loop structure with two potential CsrA binding sites located on the 5' untranslated region (UTR) of <i>ehxB</i>, which encodes a translocator required for EhxA secretion. CsrA inhibits EhxA secretion by directly binding to the RNA leader sequence of <i>ehxB</i> to repress its expression in two different ways: CsrA either binds to the Shine–Dalgarno sequence of <i>ehxB</i> to block ribosome access or to <i>ehxB</i> transcript to promote its mRNA decay. The predicted CsrA-binding site 1 of <i>ehxB</i> is essential for its regulation. There is a single potential CsrA-binding site at the 5'-end of the <i>hlyE</i> transcript, and its mutation completely abolishes CsrA-dependent activation. CsrA can also stabilize <i>hlyE</i> mRNA by directly binding to its 5' UTR. Overall, our results indicate that CsrA acts as a hemolysis modulator to regulate pathogenicity under certain conditions.

转录后全局碳存储调节因子A（Post-transcriptional global carbon storage regulator A，CsrA）是一类序列特异性RNA结合蛋白，参与调控多种细菌生理过程。溶血素（hemolysin）是肠出血性大肠杆菌（enterohemorrhagic Escherichia coli）O157:H7（EHEC）的重要毒力因子。本研究证实，CsrA在EHEC的溶血活性调控中发挥双重作用：可显著抑制质粒编码的肠溶血素（enterohemolysin，EhxA）介导的溶血过程，并通过不同机制激活染色体编码的溶血素E（hemolysin E，HlyE）介导的溶血活性。RNA结构预测结果显示，在编码EhxA分泌必需转位因子的ehxB基因的5'非翻译区（untranslated region，UTR）上，存在两个匹配度优异的茎环结构与潜在CsrA结合位点。CsrA可通过两种不同方式直接结合ehxB的RNA前导序列以抑制其表达，进而阻断EhxA的分泌：一是结合ehxB的夏因-达尔加诺（Shine–Dalgarno）序列，阻碍核糖体的结合；二是结合ehxB转录本以促进其mRNA降解。ehxB的预测CsrA结合位点1对该调控过程不可或缺。在hlyE转录本的5'端仅存在一个潜在CsrA结合位点，该位点突变可完全消除CsrA依赖的激活效应。此外，CsrA还可通过直接结合hlyE的5' UTR稳定其mRNA分子。综上，本研究结果表明，在特定条件下，CsrA可作为溶血调控因子调节病原菌的致病性。