Global post-transcriptional regulators in P. aeruginosa

摘要 铜绿假单胞菌是人类重要的条件致病菌。这是造成的主要原因 囊性纤维化 (CF) 患者的发病率和死亡率是医院获得性肺炎的主要原因， 尤其是烧伤伤口感染的问题。 Hfq 是一种保守的全球转录后调节因子 铜绿假单胞菌的毒力所必需的。在其他生物体中，Hfq 以其促进 小调节 RNA (sRNA) 与其目标转录本之间的碱基配对。 Hfq促进的相互作用 sRNA 与其 mRNA 靶标之间的连接通常起到抑制靶标翻译的作用，尽管 Hfq 可以 也以不依赖 sRNA 的方式抑制翻译。我们发现，在铜绿假单胞菌中，Hfq 共生体 从 RNA 聚合酶中产生数百个转录本。因为转录和翻译是 在细菌中，Hfq 与这些新生转录物的相互作用可能允许这种 RNA 结合 蛋白质尽早发挥其对翻译的调节作用。在目标 1 中，我们建议 鉴定与铜绿假单胞菌中 Hfq 相互作用的所有 sRNA 的靶标，并确定这些是否 sRNA 经常与 Hfq 一起作用于新生转录本。 Hfq与新生的相互作用 转录本对于如何控制转录本丰度具有重要意义；具体来说，Hfq- 新生转录本翻译的依赖性抑制将使它们易于转录 终止因子Rho或核糖核酸酶。在目标 1 中，我们将确定 Hfq 是否会降低 它通过对 Rho 介导的转录终止的影响或通过影响而结合的那些新生转录本 关于RNA降解。我们在铜绿假单胞菌中发现了第二个全球转录后调节因子，称为 RsmA 还针对数百个新生转录本，其中包括许多 Hfq 所针对的转录本。 RsmA 是 铜绿假单胞菌急性和慢性感染表型之间转换的关键调节因子，在目标 2 中我们 将调查 Hfq 和 RsmA 是否通过竞争或合作来调节彼此的活动 共同目标。与 Hfq 一样，RsmA 与目标 mRNA 种类的结合通常会抑制其翻译。在 目标 3 我们建议确定 RsmA 与新生转录本的相互作用是否影响它们 通过对 Rho 介导的转录终止的影响或对 RNA 降解的影响来丰度。的 该提案中概述的实验有望阐明 Hfq 和 RsmA 如何控制基因表达 通过他们广泛瞄准新生转录本。这些全球后转录的活动 转录调节因子尚未得到充分探索，我们提出的研究可能不仅具有影响 了解 Hfq 和 RsmA 如何影响铜绿假单胞菌的毒力，以及它们的直系同源物如何影响 含有它们的其他病原细菌中的毒力基因表达。

Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients, a major cause of hospital-acquired pneumonia and is particulary problematic in burn wound infections. Hfq is a conserved global post-transcriptional regulator that is required for the virulence of P. aeruginosa. In other organisms Hfq is best known for its ability to promote the base-pairing between small regulatory RNAs (sRNAs) and their target transcripts. Hfq-promoted interaction between an sRNA and its mRNA target typically functions to repress translation of the target, although Hfq can also inhibit translation in an sRNA-independent fashion. We have found that in P. aeruginosa Hfq associates with hundreds of transcripts as they emerge from RNA polymerase. Because transcription and translation are coupled in bacteria, the interaction of Hfq with these nascent transcripts presumably allows this RNA-binding protein to exert its regulatory effects on translation at the earliest possible opportunity. In Aim 1 we propose to identify the targets of all the sRNAs that interact with Hfq in P. aeruginosa and determine whether these sRNAs frequently work in conjunction with Hfq on nascent transcripts. The interaction of Hfq with nascent transcripts has important implications for how transcript abundance can be controlled; specifically, Hfq- dependent inhibition of the translation of nascent transcripts would render them accessible to the transcription termination factor Rho or to ribonucleases. In Aim 1 we will determine whether Hfq reduces the abundance of those nascent transcripts it binds through effects on Rho-mediated transcription termination or through effects on RNA degradation. We have found that a second global post-transcriptional regulator in P. aeruginosa called RsmA also targets hundreds of nascent transcripts, including many of those that are targeted by Hfq. RsmA is a key regulator of the switch between acute and chronic infection phenotypes in P. aeruginosa and in Aim 2 we will investigate whether Hfq and RsmA modulate each other’s activities through competition or cooperation on common targets. Like Hfq, the binding of RsmA to target mRNA species typically inhibits their translation. In Aim 3 we propose to determine whether the interaction of RsmA with nascent transcripts influences their abundance through effects on Rho-mediated transcription termination or effects on RNA degradation. The experiments outlined in this proposal are expected to illuminate how Hfq and RsmA control gene expression through their widespread targeting of nascent transcripts. The co-transcriptional activities of these global post- transcriptional regulators have been underexplored and our proposed studies may have implications not only for how Hfq and RsmA impact the virulence of P. aeruginosa, but also for how their orthologs influence virulence gene expression in other pathogenic bacteria that contain them.