Localized gene expression within P. aeruginosa biofilms

描述(由申请人提供)：许多条件致病菌可以定植于人体组织或人工植入设备，导致生物膜相关细菌感染。与在浮游培养中生长的同一菌株相比，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更强的抵抗力。这些增强阻力的机制还不是很清楚。由于大多数抗生素完全穿透微生物生物膜，生物膜感染的持久性可能涉及与更敏感的抗生素和宿主敏感的浮游生物细胞相比，生物膜相关细胞的生理差异。生物膜相关细胞和浮游细胞在转录和蛋白质组学方面的差异已经取得了进展。然而，由于在生物膜中生长的细菌不会经历相同的环境条件，因此在整个生物膜中，细胞在生理上并不相同。因此，为了了解使生物膜在传染病中持续存在的因素，识别和表征在生物膜内的空间定位位置表达的新因素是至关重要的。为了启动这些研究，我们设计了实验来表征生物膜中细菌细胞亚群的生理异质性。我们将结合激光捕获显微解剖显微镜和分子方法来识别和量化生物膜中局部位置的基因表达。特别是，我们将：(I)开发必要的分析策略来量化生物膜内细胞水平的基因表达，(Ii)表征已知在铜绿假单胞菌生物膜生长、毒力和抗生素耐药性过程中重要的基因的空间转录梯度，(Iii)开发对微生物生物膜内空间定位细胞进行全球转录转录研究所必需的方案。我们的长期目标是确定抗生物膜治疗的靶点，并了解这些靶点的局部表达如何影响细菌的毒力、抗生素耐受性和细菌在生物膜中的持久性。

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.