Biological mechanisms and consequences of efficient extracellular electron transfer in Pseudomonas aeruginosa

项目总结 铜绿假单胞菌是一种在急性感染(烧伤、伤口、呼吸机)中发现的条件致病菌 相关肺炎、眼部感染)和慢性足部感染(糖尿病溃疡)和肺部慢性感染(囊性 纤维化)。这种细菌通常以生物膜的形式存活在这些环境中，形成和高水平 对抗生素的耐受性干扰了有效的患者治疗。铜绿假单胞菌的一个决定性方面是它的 绿色素是一种有助于生物膜发育的五颜六色的氧化还原活性色素，它的制造能力及其 在感染的背景下的健康。在皮肤伤口中检测到相当浓度的绿色素。 在囊性纤维化痰中，绿青素已被证明是动物感染的毒力因子 模特们。绿青素对产生绿色素的细胞产生一系列的影响，从存在的毒性到 氧在没有氧的情况下是有益的；在氧气有限的条件下，绿色素充当电子受体。 这促进了氧化还原平衡和长期生存。这些有毒和有益的角色在 生物膜发育的不同时期，早期绿青素促进的裂解产生胞外DNA (Edna)，生物膜基质的关键成分，以及胞外多糖。最近，我们确定 Edna支持花青素在生物膜内促进细胞外电子转移(EET)的能力，促进 在氧气受限的体内的代谢活动。我们发现Edna使绿青素的保留 并将电荷转移到绿色素上。做出这些发现的关键是我们开发了新的 生物电化学技术和方法以及先进光谱技术的应用 直接探查生物膜中的EET。我们现在寻求扩展我们的跨学科工作，以获得一个机械的 了解生物膜EET效率如何通过基质的组成和结果进行调整 这可能对抗生素的耐受性有影响。某些胞外多糖(PEL、PSL)与EDNA的比率 调节绿色素在基质中的扩散系数，控制EET效率？EET效率是否与 生物膜内部的氧化还原平衡速率？绿青素介导的细胞效应，包括EET，是如何 促进生物被膜中的抗生素耐受性，这些机制是否因 微环境中的氧气？不同产绿青素的铜绿假单胞菌的相对敏感性 抗生素的分离株与其基质组成和EET效率有关？Aim1将探讨 基质组成，特别是PEL和PSL胞外多糖与EDNA的比率决定了EET 效率。目标2将检验绿青素是一种多功能的内在耐受因子的假设，其中PYO-EET 帮助铜绿假单胞菌生物膜耐受机械上不同的和临床上重要的抗生素类别 生物能量效应和/或通过诱导防御机制；我们预测主导机制 PYO影响耐受性将随着氧气浓度的不同而不同。实现这些目标将为 设计更好的控制铜绿假单胞菌生物被膜的策略所需的基础知识。

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 pyocyanin, a colorful redox-active pigment that contributes to biofilm development and its fitness in the context of infection. Pyocyanin has been detected at appreciable concentrations in skin wounds and in cystic fibrosis sputum, and pyocyanin has been shown to be a virulence factor in animal infection models. Pyocyanin exerts a range of effects over the cells that produce it, ranging from toxic in the presence of oxygen to beneficial in its absence; under oxygen-limited conditions, pyocyanin serves as an electron acceptor that promotes redox-balancing and long-term survival. These toxic and beneficial roles are important at different times in biofilm development, with early pyocyanin -promoted lysis generating extracellular DNA (eDNA), a key component of the biofilm matrix together with exopolysaccharides. Recently, we determined that eDNA underpins pyocyanin’s ability to promote extracellular electron transfer (EET) within biofilms, facilitating metabolic activity in the oxygen-limited interior. We found that eDNA enables both the retention of pyocyanin and charge transfer to pyocyanin. Critical to making these discoveries was our development of new bioelectrochemical technologies and approaches and the application of advanced spectroscopic techniques to directly probe EET in biofilms. We now seek to extend our interdisciplinary work to gain a mechanistic understanding of how biofilm EET efficiency is tuned by the composition of the matrix and the consequences this may have for antibiotic tolerance. Does the ratio of certain exopolysaccharides (Pel, Psl) to eDNA modulate pyocyanin diffusivity in the matrix, controlling EET efficiency? Does EET efficiency correlate with the rate of redox balancing in the biofilm interior? How do pyocyanin-mediated cellular effects, including EET, contribute to antibiotic tolerance in biofilms, and do these mechanisms differ according to the amount of oxygen in the microenvironment? Does the relative sensitivity of diverse pyocyanin-producing P. aeruginosa isolates to antibiotics correlate with their matrix composition and EET efficiency? Aim1 will explore how the matrix composition, particularly the ratio of Pel and Psl exopolysaccharides to eDNA, determines EET efficiency. Aim 2 will test the hypothesis that pyocyanin is a versatile intrinsic tolerance factor, where PYO-EET helps P. aeruginosa biofilms tolerate mechanistically distinct and clinically important antibiotic classes via bioenergetic effects and/or by inducing defense mechanisms; we predict the dominant mechanism by which PYO impacts tolerance will differ as a function of oxygen concentration. Attainment of these objectives will lay the foundation of basic knowledge necessary to design better strategies to control P. aeruginosa biofilms.