An essential RNA chaperone in Pseudomonas aeruginosa

摘要 铜绿假单胞菌是人类重要的条件致病菌。这是导致 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。我们 正在提议研究Hfq，一种高度保守的RNA伴侣蛋白，被认为通过其 与小调控RNA(SRNA)和mRNAs的相互作用。我们已获得证据，证明HFQ是必不可少的 在铜绿假单胞菌PAO1和含有Hfq缺失细胞中携带允许这些细胞的抑制子 为了成长。在这里，我们建议确定Hfq在铜绿假单胞菌细胞中所起的作用，并确定 Hfq在该生物体中重要性的分子基础。特别是，在目标1中，我们建议识别sRNA 以及Hfq使用RNA免疫沉淀与高通量相结合的mRNA转录本 DNA测序(RIP-Seq)。在目标2中，我们建议使用基于ClpXP的耗竭系统 用RNA-Seq来确定Hfq耗尽对全基因组范围内转录丰度的影响 铜绿假单胞菌。我们的初步RIP-Seq研究表明，HfQ与几个关键基因的转录本有关 铜绿假单胞菌毒力基因表达的调节，作为第二个目标的一部分，我们建议 确定这种关联是否可能在转录本或蛋白质水平上发挥任何调节作用 富足。在目标3中，我们将识别那些可以抑制Hfq突变影响的突变。我们会 然后尝试确定这些突变在存在和存在的情况下对基因表达的影响 没有Hfq。我们的研究有可能揭示关键的RNA伴侣的重要性的基础 在铜绿假单胞菌。由于Hfq似乎对铜绿假单胞菌的生长至关重要，我们提出的工作 可能有助于开发治疗铜绿假单胞菌患者的新疗法 感染。

Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients and a major cause of hospital-acquired pneumonia. We are proposing to study Hfq, a highly conserved RNA chaperone that is thought to function through its interactions with small regulatory RNAs (sRNAs) and mRNAs. We have obtained evidence that Hfq is essential in P. aeruginosa strain PAO1 and that cells containing a deletion of hfq carry suppressors that allow these cells to grow. Here we propose to determine what roles Hfq plays in cells of P. aeruginosa and determine the molecular basis for the essentiality of Hfq in this organism. In particular, in Aim 1 we propose to identify sRNAs and mRNA transcripts that Hfq associates with using RNA immunoprecipitation coupled with high-throughput DNA sequencing (RIP-Seq). In Aim 2 we propose to use a ClpXP protease-based depletion system coupled with RNA-Seq to determine what effect Hfq depletion has on transcript abundance on a genome-wide scale in P. aeruginosa. Our preliminary RIP-Seq studies indicate that Hfq associates with the transcripts of several key regulators of virulence gene expression in P. aeruginosa, and as part of the second aim we propose to determine whether this association might exert any regulatory effect at the level of either transcript or protein abundance. In Aim 3 we will identify those mutations that can suppress the effects of a hfq mutation. We will then attempt to determine what effect these mutations have on gene expression in both the presence and absence of Hfq. Our studies have the potential to reveal the basis for the essentiality of a key RNA chaperone in P. aeruginosa. Because Hfq appears to be critical for the growth of P. aeruginosa, the work we propose might aid in the development of novel therapeutics for the treatment of patients that suffer from P. aeruginosa infections.