Integration of redox-balancing mechanisms in Pseudomonas aeruginosa biofilms

描述（由申请人提供）：在许多类型的感染过程中，病原菌形成称为生物膜的多细胞群落。在这些聚集体中，消耗和有限的扩散导致底物可用性的陡峭梯度。微环境的建立与传统实验室液体培养的化学性质显著不同。由于生物膜中的细菌对这些条件作出反应，群落变得代谢异质，并表现出对环境扰动和抗生素治疗的抵抗力增加。虽然已知生物膜中细菌的代谢状态对于它们的耐受性是重要的，但是关于它们对底物限制的响应的原理仍然存在许多问题。我们采用菌落形态学分析，以研究生物膜的发展，在医院流行的病原菌铜绿假单胞菌。我们调节氧气和硝酸盐的可用性，这是这种细菌的已知呼吸底物，并改变铜绿假单胞菌的能力， 产生吩嗪，内源性色素，也可以作为电子受体。我们已经观察到，电子受体的可用性是生物膜结构的主要决定因素。这些研究表明，菌落生长是一种适应，当其他电子受体不可用时，允许铜绿假单胞菌细胞通过增加的表面积获得氧气。测量野生型和不能产生吩嗪的突变体中的NADH/NAD+比率表明，细胞内氧化还原状态是触发从光滑到起皱的形态型转换的信号。蛋白质组学研究和遗传筛选已经发现了候选调节剂，包括PAS结构域蛋白和与厌氧代谢和反硝化有关的调节剂，这些调节剂可能介导这种发育转变。我们的总体目标是确定生物膜中细胞的氧化还原平衡机制，确定其利用率及其在生物膜发育过程中的时空整合的条件。我们假设一个复杂的调控网络控制着铜绿假单胞菌生物膜的代谢和形态发生反应，从而维持细胞内的氧化还原稳态。我们将绘制电子受体的可用性和内部和细胞外的氧化还原电位在发展中的殖民地（目标1）。我们将验证吩嗪生物合成/还原和脱氮途径参与调控串扰，并描绘控制其在生物膜中活性的调控级联（目的2）。最后，我们将表征菌落结构测定所需的组件，并研究PAS结构域蛋白依赖的机制，连接电子受体的可用性和社区行为（目标3）。这些多条调查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域内细菌生理学的影响。铜绿假单胞菌整合环境线索以支持在拥挤结构中生长和存活的方法可能广泛适用于许多细菌病原体，并有可能为未来的治疗考虑提供信息。

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.