Localized gene expression within P. aeruginosa biofilms

描述（由申请人提供）：许多机会致病菌可定植于人体组织或人工植入器械，导致生物膜相关细菌感染。当与生长在无菌培养中的相同菌株相比时，生长在生物膜中的细菌通常对抗菌剂和宿主防御过程具有增加的抗性。这些增强的抗性的机制还没有很好地理解。由于大多数抗生素完全穿透微生物生物膜，生物膜感染的持续性可能涉及生物膜相关细胞与其更敏感的宿主敏感的生物膜对应物相比的生理差异。生物膜相关细胞与非生物膜相关细胞在转录组和蛋白质组上的差异研究已取得进展。然而，由于在生物膜中生长的细菌不经历均匀的环境条件，因此细胞在整个生物膜中在生理上是不相同的。因此，为了了解在感染性疾病中允许生物膜持续存在的因素，必须鉴定和表征在生物膜内的空间定位位点表达的新因子。为了启动这些研究，我们设计了实验来表征生物膜内细菌细胞亚群的生理异质性。我们将结合联合收割机激光捕获显微镜和分子方法，以确定和定量的基因表达在生物膜内的局部网站。我们尤其将：（i）开发在生物膜内在细胞水平上定量基因表达所必需的分析策略，（ii）表征已知在铜绿假单胞菌生物膜生长、毒力和抗生素抗性期间重要的基因的空间转录梯度，（iii）开发对微生物生物膜内的空间定位细胞进行全局转录组学研究所必需的方案。我们的长期目标是确定抗生物膜疗法的靶点，并了解这些靶点的局部表达如何影响细菌毒力，抗生素耐受性和生物膜中细菌的持久性。

DESCRIPTION (provided by applicant): Many opportunistic pathogens can colonize human tissue or artificial implant devices leading to biofilm associated bacterial infections. Bacteria growing in biofilms often have increased resistances to antimicrobials and to host defensive processes, when compared to the same strain growing in planktonic culture. The mechanisms for these enhanced resistances are not well understood. Since most antibiotics fully penetrate microbial biofilms, persistence of biofilm infections likely involves physiological differences of the biofilm-associated cells compared to their more antibiotic-sensitive and host-susceptible planktonic counterparts. Progress has been made on the transcriptomic and proteomic differences between biofilm associated and planktonic cells. However, since bacteria growing in biofilms do not experience homogenous environmental conditions, the cells are not physiologically identical throughout the biofilm. Therefore, to gain an understanding of the factors that allow biofilm persistence in infectious diseases, it is essential to identify and characterize novel factors that are expressed at spatially localized sites within biofilms. To initiate these studies, we have designed experiments to characterize the physiological heterogeneities of subsets of bacterial cells within biofilms. We will combine laser capture microdissection microscopy and molecular approaches to identify and quantify the expression of genes at localized sites within biofilms. In particular we will: (i) develop analytical strategies necessary to quantify gene expression at cellular level within biofilms, (ii) characterize spatial transcription gradients of genes known to be important during Pseudomonas aeruginosa biofilm growth, virulence, and antibiotic resistance, (iii) develop the protocols necessary to perform global transcriptomic studies on spatially localized cells within microbial bioftlms. Our long term goals are to identify targets for anti-biofilm therapies, and to understand how localized expression of these targets affect bacterial virulence, antibiotic tolerance, and persistence of bacteria in biofilms.