Biological consequences of enzymatic inactivation of Pseudomonas pyocyanin

项目摘要 铜绿假单胞菌是一种机会致病菌，在急性感染（烧伤，创伤，呼吸机 相关性肺炎、眼部感染）以及足部（糖尿病溃疡）和肺部（囊性 纤维化）。这种细菌通常以生物膜的形式在这些环境中生存，其形成和高水平的 其抗生素耐受性干扰有效的患者治疗。铜绿假单胞菌的一个定义方面是其 能够制造吩嗪，彩色氧化还原活性颜料，介导各种过程，包括 在缺氧的生物膜内部生存。像生物膜一样，CF患者肺部聚集的粘液显示出 陡峭氧气（O2）梯度。随着时间的推移，铜绿假单胞菌通常在CF中占主导地位 肺，因为它的生理允许它在这种环境中茁壮成长。随着O2的下降，吩嗪的含量上升， 某些吩嗪的浓度，如绿脓菌素（PYO）-动物感染模型中的毒力因子- 与肺功能下降有关虽然PYO是如何产生并影响不同的细胞类型的（对 生产者，和消极的主机）是众所周知的，减少PYO的潜在影响 宿主-病原体相互作用的浓度未知。最近，我们发现了一种新的PYO脱甲基酶， （PodA）由偶发分枝杆菌复合体成员产生，可感染CF患者。poda 将PYO转化为1-羟基-吩嗪（1OHPHZ），并阻断生物膜的形成和发展。PYO是 已知触发eDNA释放和促进生物膜形成，以及维持铜绿假单胞菌的厌氧 通过称为细胞外电子转移（EET）的过程进行代谢。在PYO丰富的感染中，我们 假设PodA可能通过抑制eDNA释放和消除EET来帮助控制铜绿假单胞菌， 将PYO转化为1OHPHZ。在这里，我们试图获得对PodA及其 抗生物膜活性的机制作为评估其治疗潜力的第一步。首先， PodA催化PYO去甲基化？第二，去除PYO和1OHPHZ的后果是什么？ 铜绿假单胞菌的形成第三，PodA活性是否能增强常规抗生素的有效性？ 在控制铜绿假单胞菌生长缓慢，氧气有限的生物膜区域？为了回答这些问题，我们 提出两个具体目标。目的1探讨PodA的酶活性及其作用机制 酶详细目的2探讨PodA抑制铜绿假单胞菌生物被膜的机制 在早期和晚期阶段的发展，以及它是否能使铜绿假单胞菌对妥布霉素和 环丙沙星。这些目标的实现将为评估所需的基本知识奠定基础 PodA作为治疗酶的潜在用途。

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.