Role of Non-Coding RNAs in P. aeruginosa Biofilm Development

描述（由申请方提供）：铜绿假单胞菌是一种机会致病菌，可在遗传性疾病囊性纤维化（CF）患者的肺分泌物上形成生物膜相关感染。这些感染通常不可能用传统的抗生素疗法根除。因此，研究人员对铜绿假单胞菌进行了全球转录和蛋白质组学研究，以确定感染过程中铜绿假单胞菌中诱导的新分子靶标。在许多情况下，特定蛋白质的产生在转录后水平上由小的反式作用RNA或与mRNA的5 '-非翻译区相关的顺式作用RNA调节。这些非蛋白编码RNA（ncRNA）控制许多细胞过程的表达，包括参与群体感应和毒力因子产生的过程。之前已经鉴定了大约20种铜绿假单胞菌ncRNA。根据我们的生物信息学研究，这个数字只是铜绿假单胞菌实际编码的一小部分。我们预测超过100个ncRNA编码在铜绿假单胞菌基因组的基因间区域，其他的可能被错误地注释为蛋白质编码序列。我们已经通过使用定制设计的微阵列和北方印迹实验证明了许多这些预测的ncRNA的表达。我们目前的目标是：（I）绘制铜绿假单胞菌ncRNA的基因组位置，并在具有平铺探针的定制微阵列上表示每一个。将通过使用微阵列、北方印迹和突变研究来确定这些ncRNA的表达。(II)定义位于铜绿假单胞菌基因PA 4634和PA 4635之间的新型ncRNA的生物活性。这种小的ncRNA仅在稳定期表达，此时铜绿假单胞菌缺乏铁，这种情况可能存在于感染性生物膜的内部。我们将表征这种ncRNA的表达并鉴定其mRNA靶点。我们的长期目标是确定可用于抗铜绿假单胞菌治疗的新分子靶点。这些研究将通过鉴定和表征许多在铜绿假单胞菌发病机制中重要的新的调节分子来促进这一目标。 公共卫生相关性：条件致病菌，绿脓杆菌，定植于遗传性疾病囊性纤维化患者的肺组织。这些感染通常对抗生素治疗有抗药性。本研究的目的是确定铜绿假单胞菌感染期间发生的转录后调节过程，以确定抗铜绿假单胞菌治疗的新分子靶标。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilm-associated infections on the pulmonary secretions of patients with the genetic disorder cystic fibrosis (CF). These infections are often impossible to eradicate with traditional antibiotic therapies. Therefore, investigators have performed global transcriptional and proteomics studies on P. aeruginosa to identify new molecular targets that are induced in P. aeruginosa during infectious processes. In many cases, production of specific proteins is regulated at the post-transcriptional level by small trans-acting RNAs or by cis-acting RNAs associated with the 5'-untranslated region of the mRNA. These non-protein-coding RNAs (ncRNAs) control expression of many cellular processes, including those involved in quorum sensing and virulence factor production. Approximately twenty P. aeruginosa ncRNAs have been identified previously. Based on our bioinformatics studies, this number is only a fraction of those actually encoded by P. aeruginosa. We predict that over 100 ncRNAs are encoded in the intergenic regions of the P. aeruginosa genome, and that others may be misannotated as protein-coding sequence. We have demonstrated expression of many of these predicted ncRNAs by using custom-designed microarrays and by Northern blotting experiments. Our present goals are to: (I) Map the genome position of the P. aeruginosa ncRNA, and to represent each on a custom microarray with tiled probes. Expression of these ncRNAs will be determined by using microarrays, Northern blotting, and mutational studies. (II) Define the biological activities of a novel ncRNA, located between P. aeruginosa genes PA4634 and PA4635. This small ncRNA is expressed only during stationary phase when P. aeruginosa is starved for iron, conditions likely to exist in the interiors of infectious biofilms. We will characterize the expression of this ncRNA and identify its mRNA targets. Our long-term goal is to identify new molecular targets that can be used for anti-P. aeruginosa therapies. These studies will facilitate this goal by identifying and characterizing many new regulatory molecules important in P. aeruginosa pathogenesis. PUBLIC HEALTH RELEVANCE: The opportunistic pathogen, Pseudomonas aeruginosa, colonizes pulmonary tissue of patients with the genetic disorder, cystic fibrosis. These infections are often resistant to antibiotic treatment. The goals of this study are to define post- transcriptional regulatory processes that occur during P. aeruginosa infection, in order to define novel molecular targets for anti-P. aeruginosa therapies.