The role of NosP in Pseudomonas aeruginosa biofilm development

细菌生物被膜是一种公共卫生威胁，因为它们会引起慢性和医院获得性感染，但 对抗生素有抗药性。未能表征导致生物膜扩散风险的生化机制 缺少传染病治疗的关键目标。尽管一氧化氮(NO)引发的生物膜在 铜绿假单胞菌(PA)是囊性纤维化和医院获得性感染的主要病原体，情况良好 记录在案，对相关的潜在生化过程还不了解。为了弥合这一知识 GAP，PI的长期目标是确定细菌中NO信号的机制并利用这一机制 开发驱散生物膜的治疗策略的知识。在她之前的RO1资金期，PI 建立了在她的实验室中发现的NosP(无感觉蛋白)血红素蛋白家族，作为 没有传感器来调节许多细菌中的生物膜。根据上一个资助期的研究， 拟议工作的目的是表征NOSP相关的组氨酸激酶(NAHK)并确定 NO/NOSP/NAHK调节PA生物膜和毒力的分子机制。这是假设的 RSMA下游产生的NO与NOSP结合，通过NAHK和GACS/RSM触发生物膜扩散 MKN.GACS/RSM MKN集成了来自多个传感器激活器的信号，以控制主调节器RSMA PA的运动性/急性感染与生物膜/慢性感染。这项提议具有创新性，因为它挑战了 建立了GACS/RSM MKN，并在厌氧呼吸、NO、生物膜、 和致命性。这一建议意义重大，因为阐明PA中NO信号的基础将打开新的 控制由这种重要的人类病原体引起的感染的治疗机会。这一假说将 通过追求三个具体目标进行测试：(1)确定GACS/RSM MKN中NOSP/NAHK的相互作用；(2) 描述NosP/NAHK在调节RSMA控制的表型中的作用；以及(3)建立联系 RSMA调节的反硝化作用与NOSP/NAHK信号之间的关系。在目标1下，NAHK蛋白质和 与Gacs/RSM MKN成员的磷转移相互作用将被表征为NO/NOSP的函数 监管。在目标2下，NO/NOSP/NAHK对毒力、群体感应和环二GMP途径的影响 由RSMA控制的将被量化。在目标3下，NO/NOSP/NAHK对RSMA控制的影响 反硝化将作为一个监管反馈回路进行量化和调查。私家侦探有丰富的经验 与建议的化验结果一致。在完成这些目标后，预计将在#年设立NO/NOSP/NAHK 控制Gacs/RSM动力/毒力开关。这将是一个根本性的重要发现， 确定新的信号通路和新的抗生素靶点，这是迫切需要的，特别是 考虑到在生物膜生物中典型的抗生素耐药性增加。非触发生物膜 在细菌中广泛观察到了扩散，因此基于NO信号的临床干预具有潜在的可能性。 对于广泛的应用，进一步加深了本项目的意义。

Bacterial biofilms are a public health threat because they cause chronic and hospital-acquired infections but are resistant to antibiotics. Failure to characterize the biochemical machinery that drives biofilm dispersal risks missing key targets for treatment of infectious disease. Although nitric oxide (NO)-triggered biofilm dispersal in Pseudomonas aeruginosa (PA), a principal pathogen in cystic fibrosis and hospital-acquired infections, is well documented, the underlying biochemical processes responsible are not understood. To bridge this knowledge gap, a long-term goal of the PI is to determine the mechanism of NO signaling in bacteria and to use this knowledge to develop therapeutic strategies to disperse biofilms. In her previous RO1 funding period, the PI established the NosP (NO sensing protein) family of hemoproteins, which were discovered in her laboratory, as NO sensors that regulate biofilm in many bacteria. Based on studies from the previous funding period, the objective of the proposed work is to characterize the NosP-associated histidine kinase (NahK) and determine the molecular mechanism underlying NO/NosP/NahK regulation of biofilm and virulence in PA. It is hypothesized that NO produced downstream of RsmA binds NosP to trigger biofilm dispersal through NahK and the GacS/Rsm MKN. The GacS/Rsm MKN integrates signals from many sensor kinases to control RsmA, the master regulator of motility/acute infection v. biofilm/chronic infection in PA. The proposal is innovative because it challenges the established GacS/Rsm MKN and forges new logical connections between anaerobic respiration, NO, biofilm, and virulence. This proposal is significant because elucidation of the basis for NO signaling in PA will open new therapeutic opportunities for controlling infection caused by this important human pathogen. The hypothesis will be tested by pursuing three specific aims: (1) to identify NosP/NahK interactions within the GacS/Rsm MKN; (2) to delineate the role of NosP/NahK in modulation of RsmA-controlled phenotypes; and (3) to establish the link between RsmA-regulated denitrification and NosP/NahK signaling. Under aim 1, NahK protein-protein and phosphotransfer interactions with GacS/Rsm MKN members will be characterized as a function of NO/NosP regulation. Under aim 2, the effect of NO/NosP/NahK on virulence, quorum sensing, and cyclic-di-GMP pathways controlled by the RsmA will be quantified. Under aim 3, the effect of NO/NosP/NahK on RsmA-controlled denitrification will be quantified and investigated as a regulatory feedback loop. The PI has significant experience with the proposed assays. Upon completion of these aims, NO/NosP/NahK is expected to be established in controlling the GacS/Rsm motility/virulence switch. This would represent a fundamentally important discovery, defining a new signaling pathway and novel antibiotic targets, for which there is a pressing need, especially considering the increased antibiotic resistance typically seen in biofilming organisms. NO-triggered biofilm dispersal has been widely observed in bacteria, so clinical interventions based on NO signaling have the potential for widespread application, furthering the significance of this project.