Integration of redox-balancing mechanisms in Pseudomonas aeruginosa biofilms

铜绿假单胞菌生物膜中氧化还原平衡机制的整合

基本信息

批准号：
8577604
负责人：
Lars Dietrich
金额：
$ 37.22万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-20 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8577604
关键词：
Adopted Affect Agar Anabolism Antibiotic Therapy Area Bacteria Bacterial Physiology Behavior Biological Assay Cells Chemistry Chimeric Proteins Chronic Communities Complex Consumption Crowding Cues Cystic Fibrosis Development Diffusion Disease Electrodes Electrophoretic Mobility Shift Assay Energy Metabolism Equilibrium Exhibits Fluorescence Microscopy Future Gene Expression Gene Expression Profile Genetic Screening Goals Growth Homeostasis Hospitals Hypoxia Image In Situ In Vitro Individual Infection Laboratories Life Style Link Liquid substance Lung Maintenance Maps Measurement Mediating Metabolic Metabolic Control Metabolism Microbial Biofilms Microelectrodes Microscopy Microtomy Modeling Molecular Molecular Profiling Monitor Morphogenesis Morphology NADH NBL1 gene Nitrates Oxidants Oxidation-Reduction Oxygen Pathway interactions Patients Phenazines Photons Physiology Pigments Production Proteomics Pseudomonas aeruginosa Quinolones Reaction Regulation Regulon Reporter Resistance Role Signal Transduction Structure Surface Techniques Tertiary Protein Structure Testing Therapeutic Time Variant Work appendage base cell motility coping denitrification experience extracellular fimbria genetic regulatory protein knowledge base microbial mortality mutant pathogen pathogenic bacteria protein activation public health relevance respiratory response spatiotemporal therapy resistant transcription factor

项目摘要

DESCRIPTION (provided by applicant): During many types of infections, pathogenic bacteria form multicellular communities called biofilms. In these aggregates, consumption and limited diffusion leads to steep gradients of substrate availability. Microenvironments are established that differ significantly from the chemistries of traditional laboratory liquid cultures. As bactera in biofilms respond to these conditions, the community becomes metabolically heterogeneous and exhibits increased resistance to environmental perturbations and antibiotic treatment. Although the metabolic states of bacteria in biofilms are known to be important for their recalcitrance, many questions remain regarding the principles that underlie their response to substrate limitation. We employ a colony morphology assay to study biofilm development in the prevalent nosocomial pathogen Pseudomonas aeruginosa. We modulate the availability of oxygen and nitrate, known respiratory substrates for this bacterium, and alter the ability of P. aeruginosa to produce phenazines, endogenous pigments that can also act as electron acceptors. We have observed that electron acceptor availability is a major determinant of biofilm structure. These studies suggest that colony wrinkling is an adaptation that allows P. aeruginosa cells to access oxygen through an increased surface area when other electron acceptors are not available. Measurement of the NADH/NAD+ ratio in the wild type and a mutant unable to produce phenazines has indicated that the intracellular redox state is a signal that triggers the morphotypic switch from smooth to wrinkled. Proteomic studies and genetic screens have uncovered candidate regulators, including PAS domain proteins and regulators implicated in anaerobic metabolism and denitrification that likely mediate this developmental transition. Our overall goal is to define the mechanisms underlying redox balancing for cells in biofilms, the conditions that determine their utilization and their spatiotemporal integration during biofilm development. We hypothesize that a complex regulatory network controls metabolic and morphogenetic responses to the conditions in P. aeruginosa biofilms such that intracellular redox homeostasis is maintained. We will map electron acceptor availability and intra- and extracellular redox potentials in developing colonies (Aim 1). We will verify that phenazine biosynthesis/ reduction and denitrification pathways engage in regulatory cross-talk and delineate the regulatory cascades controlling their activity in biofilms (Aim 2). Finally, we will characterize the components required for colony structure determination and investigate PAS domain protein-dependent mechanisms that link electron acceptor availability and community behavior (Aim 3). These multiple lines of inquiry will reveal the 3D distribution of exogenous and endogenous electron acceptors and their effects on bacterial physiology within specific microdomains of P. aeruginosa colonies. The means by which P. aeruginosa integrates environmental cues to support growth and survival in a crowded structure may be broadly applicable to many bacterial pathogens and have the potential to inform future therapeutic considerations.

描述（由申请人提供）：在许多类型的感染中，致病细菌形成了称为生物膜的多细胞群落。在这些聚集体中，消耗和有限的扩散会导致底物可用性的陡峭梯度。建立了与传统实验室液体培养物的化学物质明显不同的微环境。随着生物膜中的细菌对这些疾病的反应，社区在代谢上变得异质性，并表现出对环境扰动和抗生素治疗的耐药性。尽管众所周知，生物膜中细菌的代谢状态对于它们的顽固性很重要，但关于其对底物限制的反应的原理仍然存在许多问题。我们采用一个菌落形态测定法研究了铜绿假单胞菌流行的生物膜发育。我们调节该细菌的氧气和硝酸盐的可用性，已知的呼吸道底物，并改变了铜绿假单胞菌的能力产生促苯胺，内源性色素，也可以充当电子受体。我们已经观察到电子受体可用性是生物膜结构的主要决定因素。这些研究表明，菌落皱纹是一种适应性的适应性，可在没有其他电子受体的情况下通过增加的表面积进入氧气。在野生型中NADH/NAD+比率的测量和无法产生促苯胺的突变体的测量表明，细胞内氧化还原态是触发从光滑到皱纹的形态型开关的信号。蛋白质组学研究和遗传筛选具有捕获的候选调节剂，包括PAS结构域蛋白质和与厌氧代谢和反硝化有关的调节剂，可能介导这种发育过渡。我们的总体目标是定义生物膜中细胞的氧化还原平衡的机制，确定其利用率及其在生物膜发育过程中时空整合的条件。我们假设一个复杂的调节网络控制对铜绿假单胞菌生物膜中条件的代谢和形态发生反应，从而维持细胞内氧化还原稳态。我们将在发展菌落中绘制电子受体的可用性以及细胞内和细胞外氧化还原电位（AIM 1）。我们将验证苯嗪生物合成/还原和反硝化途径参与调节性交叉对话，并描述控制其在生物膜中活性的调节级联（AIM 2）。最后，我们将表征菌落结构确定所需的组件，并研究将电子受体可用性和社区行为联系起来的PAS结构域蛋白质依赖性机制（AIM 3）。这些多种探究线将揭示外源性和内源性电子受体的3D分布及其对铜绿假单胞菌菌落特定微区域内细菌生理的影响。铜绿假单胞菌整合环境线索以支持拥挤结构的生长和生存的手段可能广泛地适用于许多细菌病原体，并有可能为未来的治疗方面的考虑提供信息。