Integration of redox-balancing mechanisms in Pseudomonas aeruginosa biofilms

描述(申请人提供)：在许多类型的感染期间，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利用率的陡峭梯度。建立的微环境与传统实验室液体培养的化学成分有很大不同。当生物膜中的细菌对这些条件做出反应时，群落在代谢上变得异质性，并对环境扰动和抗生素治疗表现出更强的抵抗力。尽管细菌在生物膜中的代谢状态对它们的顽固性很重要，但关于它们对底物限制的反应所依据的原则，仍然存在许多问题。我们采用菌落形态分析的方法来研究医院内流行的铜绿假单胞菌的生物被膜发育。我们调节氧气和硝酸盐的可用性，这是已知的这种细菌的呼吸底物，并改变铜绿假单胞菌 产生吩嗪，这是一种内源性色素，也可以作为电子受体。我们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，菌落皱缩是一种适应，允许铜绿假单胞菌细胞在没有其他电子受体的情况下通过增加的表面积获得氧气。对野生型和不能产生吩嗪的突变体的NADH/NAD+比率的测量表明，细胞内的氧化还原状态是触发从光滑到皱纹的形态转换的信号。蛋白质组学研究和遗传筛选已经发现了候选调节因子，包括PAS结构域蛋白和参与厌氧代谢和反硝化的调节因子，它们可能介导了这种发育过渡。我们的总体目标是确定生物膜中细胞氧化还原平衡的潜在机制，决定它们的利用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对条件的代谢和形态发生反应，从而维持细胞内氧化还原动态平衡。我们将绘制发育中菌落的电子受体可用性和细胞内和胞外氧化还原电位图(目标1)。我们将验证吩嗪的生物合成/还原和反硝化途径参与调节串扰，并描绘控制其在生物膜中的活性的调节级联(目标2)。最后，我们将表征菌落结构确定所需的成分，并研究PAS结构域蛋白质依赖的机制，将电子受体的可用性和社区行为联系起来(目标3)。这多条线索将揭示外源性和内源性电子受体在铜绿假单胞菌特定微区内的三维分布及其对细菌生理的影响。铜绿假单胞菌整合环境线索以支持在拥挤的结构中生长和生存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参考。

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.