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。这是主要原因 囊性纤维化（CF）患者的发病率和死亡率以及医院获得性肺炎的主要原因。我们 正在提议研究Hfq，这是一种高度保守的RNA分子伴侣，被认为是通过其 与小调控RNA（sRNA）和mRNA的相互作用。我们已经获得证据表明Hfq是必不可少的 在铜绿假单胞菌菌株PAO 1和含有hfq缺失细胞中，携带抑制子， 生长.在这里，我们建议确定Hfq在铜绿假单胞菌细胞中起什么作用，并确定 这种生物体中Hfq的重要性的分子基础。特别地，在目标1中，我们提出鉴定sRNA 使用RNA免疫沉淀结合高通量检测与Hfq相关的mRNA转录物 DNA测序（RIP-Seq）。在目的2中，我们提出使用基于ClpXP蛋白酶的耗尽系统偶联 使用RNA-Seq来确定Hfq缺失在全基因组范围内对转录本丰度的影响， P.铜绿。我们初步的RIP-Seq研究表明，Hfq与几个关键的转录本相关， 铜绿假单胞菌中毒力基因表达的调节因子，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 丰饶。在目标3中，我们将确定那些可以抑制hfq突变影响的突变。我们将 然后试图确定这些突变对基因表达的影响， 没有HFQ。我们的研究有可能揭示一个关键RNA伴侣的重要性的基础 在铜绿假单胞菌中。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 可能有助于开发用于治疗患有铜绿假单胞菌的患者的新疗法 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.