Biological consequences of enzymatic inactivation of Pseudomonas pyocyanin

绿脓杆菌酶灭活的生物学后果

• 批准号: 9384435
• 负责人: Dianne K Newman
• 金额: $ 54.02万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-08 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9384435
• 关键词: Active Sites; Acute; Anaerobic Bacteria; Animals; Antibiotic susceptibility; Antibiotics; Bacteria; Biochemical; Biological; Biological Assay; Burn injury; Cells; Chronic; Ciprofloxacin; Collaborations; Complex; Crystallization; Cystic Fibrosis; Cytolysis; Development; Diabetic ulcer; District of Columbia; Effectiveness; Electron Transport; Environment; Enzymes; Excision; Exhibits; Eye Infections; Formaldehyde; Foundations; Future; Generations; Genus Mycobacterium; Growth; Homeostasis; Human; Hydrogen Bonding; Infection; Iron; Knowledge; Learning; Lung; Mediating; Metabolic; Metabolism; Microbe; Microbial Biofilms; Modeling; Mucociliary Clearance; Mucolytics; Mucous body substance; Mycobacterium fortuitum; Organism; Oxidants; Oxidation-Reduction; Oxygen; Patients; Phase; Phenazines; Physiologic tolerance; Physiology; Pigments; Population; Process; Pseudomonas; Pseudomonas aeruginosa; Pulmonary Fibrosis; Pyocyanine; Reaction; Reactive Oxygen Species; Research; Resolution; Respiratory physiology; Signal Transduction; Site-Directed Mutagenesis; Soil; Solvents; Specificity; Sputum; Structure; System; Testing; Therapeutic; Time; Tobramycin; Virulence Factors; Water; Work; cell type; cystic fibrosis airway; cystic fibrosis patients; demethylation; experimental study; extracellular; foot; improved; inhibitor/antagonist; member; microbial; novel; pathogen; therapeutic enzyme; treatment strategy; ventilator-associated pneumonia

PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen found in acute infections (burns, wounds, ventilator associated pneumonia, eye infections) and chronic infections of the foot (diabetic ulcers) and lung (cystic fibrosis). This bacterium commonly survives in these contexts as a biofilm, the formation and high-level antibiotic tolerance of which interferes with effective patient treatment. A defining aspect of P. aeruginosa is its ability to make phenazines, colorful redox-active pigments that mediate a variety of processes, including survival within the anoxic interior of biofilms. Like biofilms, mucus collecting in the lungs of CF patients exhibits steep oxygen (O2) gradients. Over time, P. aeruginosa commonly dominates the microbial population in the CF lung, as its physiology permits it to thrive in this environment. As O2 declines, phenazines rise, and the concentration of certain phenazines, such as pyocyanin (PYO)—a virulence factor in animal infection models— is correlated with declining lung function. While how PYO is made and impacts diverse cell types (positively for the producer, and negatively for the host) is well understood, the potential impact of reducing PYO concentration for host-pathogen interactions is unknown. Recently, we discovered a novel PYO demethylase (PodA) made by members of the Mycobacterium fortuitum complex, which can infect CF patients. PodA converts PYO to 1-hydroxy-phenazine (1OHPHZ), and blocks biofilm formation and development. PYO is known to trigger eDNA release and promote biofilm formation, as well as sustain P. aeruginosa's anaerobic metabolism via a process called extracellular electron transfer (EET). In infections where PYO is abundant, we hypothesize that PodA might help control P. aeruginosa by inhibiting eDNA release and abrogating EET by converting PYO to 1OHPHZ. Here, we seek to gain a fundamental scientific understanding of PodA and its mechanism of anti-biofilm activity as a first step towards evaluating its therapeutic potential. First, how does PodA catalyze PYO demethylation? Second, what is the consequence of PYO removal and 1OHPHZ formation for P. aeruginosa? Third, might PodA activity potentiate the effectiveness of conventional antibiotics in controlling P. aeruginosa in slowly-growing, O2-limited biofilm regions? To answer these questions, we propose two specific aims. Aim 1 will explore the enzymatic activity and mechanism of action of the PodA enzyme in detail. Aim 2 will probe the mechanisms underpinning PodA's inhibition of P. aeruginosa biofilm development at early and late stages, and whether it can sensitize P. aeruginosa to tobramycin and ciprofloxacin. Attainment of these objectives will lay the foundation of basic knowledge necessary to evaluate the potential usage of PodA as a therapeutic enzyme.

项目摘要 铜绿假单胞菌是在急性感染中发现的机会性病原体（烧伤，伤口，呼吸机 相关的肺炎，眼部感染）和脚部（糖尿病性溃疡）和肺（囊性）的慢性感染 纤维化）。这种细菌通常在这些情况下以生物膜，形成和高级生存生存 抗生素耐受性会干扰有效的患者治疗。铜绿假单胞菌的一个定义方面是 能够制作苯胺的能力，介导各种过程的五颜六色的氧化还原活性色素，包括 生物膜缺氧内部内的生存。像生物膜一样，在CF患者的肺中收集的粘液表现出 陡峭的氧（O2）梯度。随着时间的流逝，铜绿假单胞菌通常主导CF中的微生物种群 肺部的生理学允许在这种环境中壮成长。随着O2的下降，苯丙胺的上升和 某些苯丙胺的浓度，例如上氨酸（PYO） - 动物感染模型中的病毒因子 - 与肺功能下降相关。而如何制作Pyo并影响各种细胞类型（对 生产者对宿主的负面理解，对宿主有否定的理解，减少Pyo的潜在影响 宿主 - 病原体相互作用的浓度尚不清楚。最近，我们发现了一种新型的脱甲基酶 （PODA）由Fortuitum Complex成员制造，该复合物可以感染CF患者。 poda 将PYO转换为1-羟基苯丙胺（1OHPHZ），并阻止生物膜的形成和发育。 Pyo是 已知会触发Edna释放并促进生物膜形成，以及铜绿假单胞菌的厌氧。 通过称为细胞外电子转移（EET）的过程代谢。在Pyo丰富的感染中，我们 假设PODA可以通过抑制Edna释放并通过 将PYO转换为1OHPHz。在这里，我们试图获得对Poda及其IT的基本科学理解 抗生物膜活性的机理是评估其治疗潜力的第一步。首先，如何 PODA催化Pyo脱甲基化？其次，PYO去除和1OHPHZ的结果是什么 铜绿假单胞菌的形成？第三，可能活性潜力的常规抗生素有效性 在缓慢生长的O2限制生物膜区域中控制铜绿假单胞菌？要回答这些问题，我们 提案两个具体的目标。 AIM 1将探索PODA的酶活性和作用机理 详细酶。 AIM 2将探测Poda抑制铜绿假单胞菌生物膜的抑制的机制 在早期和晚期的发展，以及是否可以将铜绿假单胞菌感知到毒素和 环丙沙星。实现这些目标将奠定评估所必需的基本知识的基础 PODA作为治疗酶的潜在用法。