Resuscitation of P. aeruginosa biofilm cells from dormancy

描述（由申请人提供）：铜绿假单胞菌是一种机会性病原体，可在囊性纤维化（CF）患者的肺组织上形成生物膜，导致慢性感染。从CF患者个体中分离的铜绿假单胞菌的纵向基因组学研究表明，随着时间的推移，细菌菌株往往是克隆的。这表明原始创始菌株的亚群在抗生素治疗中存活下来，然后在治疗减轻时重新填充生物膜。存活下来的细菌被称为持久性细胞。持久性细胞可能处于代谢休眠状态，部分原因是生物膜中含有低氧或低营养物质的微环境。为了消除慢性感染，有必要防止持久性细胞的形成或防止它们从休眠中复苏。在这里，我们将重点关注后一种方法，通过描述允许细菌在休眠条件下延长生存的因素的作用。利用激光捕获显微解剖（LCM）和转录组学，我们在铜绿假单胞菌生物膜的休眠、耐抗生素亚群中发现了丰富的mRNA转录物。这些转录本编码核糖体冬眠因子，HPF和RMF，它们在大肠杆菌中被证明可以将活性核糖体转化为静止的1000s二聚体。由于许多抗生素靶向翻译，核糖体失活的细菌对这些抗生素具有耐受性。此外，我们的初步研究表明，铜绿假单胞菌缺乏这些因素会导致在非生长条件下细胞活力的丧失。HPF的缺失导致50S核糖体亚基的23S rRNA几乎完全降解。由于现成的核糖体是细胞从休眠中恢复所必需的，这些因素可能进化为保护核糖体在饥饿条件下免于完全降解。因此，本研究的目的是进一步表征这些核糖体冬眠因子的表达和活性，并确定它们在铜绿假单胞菌生物膜细胞从休眠中恢复中的作用。在这项研究中，我们将：(i)表征RMF和HPF的分子活性，并确定它们在铜绿假单胞菌休眠生物膜亚群核糖体丰度和存活中的作用；（ii）表征RMF和HPF的转录调控，以确定它们在生物膜中高表达所需的因素；（iii）确定mRNA折叠和其他转录后调控过程在这些因子在生物膜中的表达中的作用。最终,我们

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on the pulmonary tissue of patients with cystic fibrosis (CF), resulting in chronic infections. Longitudinal genomics studies of P. aeruginosa isolates from individual CF patients indicate that the bacterial strains are often clonal over time. This suggest that subpopulations of the original founder strains survive the antibiotic treatments, then repopulate the biofilms when treatments are alleviated. The surviving bacteria are termed persister cells. Persister cells may be metabolically dormant, and arise in part because biofilms contain microenvironments with low oxygen or nutrient availability. In order to eliminate chronic infections, it will be necessary to either prevent the formation of persister cells or prevent thei resuscitation from dormancy. Here, we will focus on the latter approach, by characterizing the role of factors that allow prolonged survival of bacteria under dormant conditions. Using laser capture microdissection (LCM) and transcriptomics, we identified mRNA transcripts that are abundant in the dormant, antibiotic-tolerant subpopulations of P. aeruginosa biofilms. These transcripts code for ribosome hibernation factors, HPF and RMF, which have been shown in Escherichia coli to convert active ribosomes to resting 100S dimers. Since many antibiotics target translation, bacteria with inactivated ribosomes would be tolerant to these antibiotics. In addition, our preliminary studies demonstrate that the absence of these factors in P. aeruginosa results in loss of cell viability during non- growth conditions. Loss of HPF results in almost complete degradation of the 23S rRNA of the 50S ribosomal subunit. Since ready-made ribosomes are necessary for cell recovery from dormancy, these factors likely evolved to protect ribosomes from complete degradation during starvation conditions. Therefore, the goals of this research are to characterize further the expression and activity of these ribosome hibernation factors and determine their role in recovery of P. aeruginosa biofilm cells from dormancy. In this research we will: (i) characterize the molecular activities of RMF and HPF, and determine their role in ribosome abundances and survival of the P. aeruginosa dormant biofilm subpopulations, (ii) characterize the transcriptional regulation of rmf and hpf, to identify factors required for teir high expression in biofilms, and (iii) determine the role of mRNA folding and other post-transcriptional regulatory processes in the expression of these factors in biofilms. Ultimately, we will identify molecular mechanisms that allow recovery of biofilm cells from dormancy. These mechanisms may then be used as targets to kill the dormant biofilm bacteria that cause chronic P. aeruginosa pulmonary infections.