Mey Alexandra R, Midgett Charles R, Kull F Jon, Payne Shelley M
Department of Molecular Biosciences and LaMontagne Center for Infectious Diseases, The University of Texas at Austin, Austin, Texas, USA.
Department of Chemistry, Dartmouth College, Hanover, New Hampshire, USA.
mBio. 2024 Dec 11;15(12):e0285324. doi: 10.1128/mbio.02853-24. Epub 2024 Nov 18.
Intestinal colonization and virulence factor production in response to environmental cues is mediated through several regulatory factors in , including the highly conserved RNA-binding global regulatory protein CsrA. We have shown previously that CsrA increases synthesis of the virulence-associated transcription factor ToxR in response to specific amino acids (NRES) and is required for the virulence of in the infant mouse model of cholera. In this study, we mapped the 5' untranslated region (5' UTR) of and showed that CsrA can bind directly to an RNA sequence encompassing the 5' UTR, indicating that the regulation of ToxR levels by CsrA is direct. Consistent with this observation, the 5' UTR of contains multiple putative CsrA binding sequences (GGA motifs), and mutating these motifs disrupted the CsrA-mediated increase in ToxR. Optimal binding of CsrA to a defined RNA oligonucleotide required the bridging of two GGA motifs within a single RNA strand. To determine the mechanism of regulation by CsrA, we assayed transcript levels, stability, and efficiency of translation. Both the amount of mRNA in NRES and the stability of the transcript were increased by CsrA. Using an translation assay, we further showed that synthesis of ToxR was greatly enhanced in the presence of purified CsrA, suggesting a direct role for CsrA in the translation of mRNA. We propose a model in which CsrA binding to the 5' UTR of the transcript promotes ribosomal access while precluding interactions with RNA-degrading enzymes.IMPORTANCE is uniquely adapted to marine environments as well as the human intestinal tract. Global regulators, such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates the synthesis of ToxR, a key virulence factor of . Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization in the human host, the nutrient-rich small intestinal mucosa.
对环境信号作出反应时，肠道定殖和毒力因子产生是由多种调控因子介导的，包括高度保守的RNA结合全局调控蛋白CsrA。我们之前已经表明，CsrA在对特定氨基酸（NRES）作出反应时会增加毒力相关转录因子ToxR的合成，并且在婴儿霍乱小鼠模型中对霍乱弧菌的毒力是必需的。在这项研究中，我们绘制了霍乱弧菌的5'非翻译区（5'UTR）图谱，并表明CsrA可以直接结合到包含5'UTR的RNA序列上，这表明CsrA对ToxR水平的调控是直接的。与这一观察结果一致，霍乱弧菌的5'UTR包含多个假定的CsrA结合序列（GGA基序），突变这些基序会破坏CsrA介导的ToxR增加。CsrA与特定RNA寡核苷酸的最佳结合需要在单链RNA内桥接两个GGA基序。为了确定CsrA的调控机制，我们检测了霍乱弧菌转录水平、稳定性和翻译效率。CsrA增加了NRES中霍乱弧菌mRNA的量以及霍乱弧菌转录本的稳定性。使用霍乱弧菌翻译试验，我们进一步表明，在纯化的CsrA存在下，ToxR的合成大大增强，这表明CsrA在霍乱弧菌mRNA的翻译中起直接作用。我们提出了一个模型，其中CsrA与霍乱弧菌转录本的5'UTR结合促进核糖体进入，同时阻止与RNA降解酶的相互作用。重要性霍乱弧菌独特地适应海洋环境以及人类肠道。全局调控因子，如CsrA，有助于将环境信号转化为适当的细胞反应，对于在这些不同环境之间切换至关重要。了解传递环境信号所涉及的途径对于理解这种毁灭性人类病原体的环境持久性和肠道发病机制至关重要。在这项研究中，我们证明CsrA直接调控霍乱弧菌的关键毒力因子ToxR的合成。在有利于细胞中高水平活性CsrA的条件下，例如在特定氨基酸存在的情况下，CsrA通过结合霍乱弧菌转录本并增强其稳定性和翻译来增加ToxR蛋白水平。通过响应营养可用性，CsrA完全有能力在人类宿主中霍乱弧菌的首选定殖部位，即营养丰富的小肠粘膜，激活毒力基因调控级联反应。
