Global post-transcriptional regulators in P. aeruginosa

铜绿假单胞菌的全局转录后调节因子

• 批准号: 10524023
• 负责人: SIMON L DOVE
• 金额: $ 57.91万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-02 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524023
• 关键词: Bacteria; Base Pairing; Binding; Chronic; Coupled; DNA-Directed RNA Polymerase; Enzymes; Escherichia coli; Family; Free Ribosome; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Gram-Negative Bacteria; Infection; Licensing; Lung infections; Mediating; Messenger RNA; Molecular Chaperones; Morbidity - disease rate; Nosocomial pneumonia; Organism; Orthologous Gene; Patients; Phenotype; Prevention; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; RNA; RNA Degradation; RNA-Binding Proteins; Rho Factor; Ribonucleases; Sepsis; Stretching; Testing; Transcript; Translational Repression; Translations; Ventilator; Virulence; Work; Wound Infection; acute infection; burn wound; chronic infection; cystic fibrosis patients; experimental study; human pathogen; member; mortality; new therapeutic target; opportunistic pathogen; pathogenic bacteria; posttranscriptional; protein protein interaction; recruit; rho; ribonuclease E; transcription termination; virulence gene

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.

摘要 铜绿假单胞菌是人类重要的条件致病菌。这是主要原因 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， 尤其是在烧伤伤口感染方面存在问题。HFQ是一种保守的全局转录后调节因子， 是铜绿假单胞菌毒力所必需的。在其他生物体中，Hfq最为人所知的是其促进细胞增殖的能力。 小调节RNA（sRNA）和它们的靶转录物之间的碱基配对。HFQ促进的相互作用 sRNA与其mRNA靶之间的相互作用通常用于抑制靶的翻译，尽管Hfq可以 也以不依赖于siRNA的方式抑制翻译。我们发现在铜绿假单胞菌中， 在RNA聚合酶的作用下有数百个转录本。因为转录和翻译 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 蛋白质发挥其调节作用的翻译在尽可能早的机会。在目标1中，我们建议 鉴定铜绿假单胞菌中与Hfq相互作用的所有sRNA的靶标，并确定这些sRNA是否 sRNA经常与Hfq一起作用于新生转录物。Hfq与新生 转录本对于如何控制转录本丰度具有重要意义;具体而言，Hfq- 依赖性抑制新生转录物的翻译将使它们易于转录 终止因子Rho或核糖核酸酶。在目标1中，我们将确定Hfq是否减少了 它通过影响Rho介导的转录终止或通过影响 RNA降解。我们发现铜绿假单胞菌中的第二个全局转录后调节因子， RsmA还靶向数百种新生转录物，包括Hfq靶向的许多转录物。RsmA是 在铜绿假单胞菌和目标2中， 将研究Hfq和RsmA是否通过竞争或合作来调节彼此的活动， 共同的目标。与Hfq一样，RsmA与靶mRNA种类的结合通常抑制它们的翻译。在 目的3：确定RsmA与新生转录本的相互作用是否影响它们的表达。 通过对Rho介导的转录终止的影响或对RNA降解的影响来增加丰度。的 在这项计划中概述的实验有望阐明Hfq和RsmA如何控制基因表达 通过他们对新生转录本的广泛瞄准。这些全球性的后转录活动， 转录调节因子的研究还不够深入，我们提出的研究可能不仅会产生影响， 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 毒力基因在其他含有它们的病原菌中的表达。