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

描述(申请人提供)：铜绿假单胞菌是一种条件致病菌，在遗传性疾病囊性纤维化(CF)患者的肺部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，以确定在感染过程中诱导的新的分子靶点。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。这些非蛋白质编码的RNA(NcRNAs)控制着许多细胞过程的表达，包括那些参与群体感应和毒力因子产生的过程。以前已经鉴定了大约20个铜绿假单胞菌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数字的一小部分。我们预测在铜绿假单胞菌基因组的基因间区有100多个ncRNA被编码，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Northern杂交实验证明了许多预测的ncRNAs的表达。我们目前的目标是：(I)绘制铜绿假单胞菌ncRNA的基因组位置图，并在定制的带有平铺探针的微阵列上表示每个位置。这些ncRNAs的表达将通过微阵列、Northern印迹和突变研究来确定。(Ii)确定位于铜绿假单胞菌基因PA4634和PA4635之间的新的ncRNA的生物学活性。这种小的ncRNA只在铜绿假单胞菌缺乏铁的稳定期表达，这种情况很可能存在于感染性生物膜的内部。我们将对这种ncRNA的表达进行鉴定，并确定其mRNA靶标。我们的长期目标是寻找新的可用于抗肺炎支原体的分子靶点。铜绿假手术疗法。这些研究将通过识别和表征许多在铜绿假单胞菌发病机制中重要的新调节分子来促进这一目标。 公共卫生相关性：条件致病菌铜绿假单胞菌定植于患有遗传性疾病囊性纤维化的患者的肺组织。这些感染往往对抗生素治疗产生抗药性。本研究的目标是确定在铜绿假单胞菌感染过程中发生的转录后调控过程，以确定新的抗铜绿假单胞菌分子靶点。铜绿假手术疗法。

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.