The role of NosP in Pseudomonas aeruginosa biofilm development

细菌生物膜是相当大的公共卫生威胁，因为它们引起慢性和医院获得性感染。 感染，以及医疗植入物的持续生物污垢，但对抗生素有抗药性。生物膜 已经在细菌中广泛观察到NO的调节，因此基于NO信号传导的治疗干预 会对公众健康产生重大影响然而，人们对细菌NO信号传导的了解却很少。为 例如，NO调节铜绿假单胞菌中的生物膜扩散，铜绿假单胞菌是囊性纤维化的主要病原体， 医院获得性感染，是非常有据可查的，但NO传感器仍然未知。以弥补这一 知识差距，PI的长期目标是确定细菌中NO信号传导的机制， 使用这一知识作为开发治疗策略以分散生物膜的基础。主要研究者有 显示NO调节许多细菌（包括铜绿假单胞菌）的生物膜形成，但铜绿假单胞菌缺乏一种 H-NOX蛋白的同源物显示在其他物种中介导对NO的反应。因此，铜绿假单胞菌 必须有备用NO传感器。PI发现了一个新的血红素蛋白家族，名为NosP。基于 根据强有力的初步数据，假设NosP是调节生物膜形成的NO传感器。的 拟议工作的目的是表征铜绿假单胞菌NosP，并确定该蛋白在 NO介导的生物膜形成的控制。这一建议是创新的，因为它建立了新的逻辑联系 NO传感和生物膜形成之间的关系，为NO在细菌中的作用建立了一个新的范例。这 这一提议是重要的，因为阐明铜绿假单胞菌中NO信号传导的基础将打开新的 控制由这种重要的人类病原体引起的感染的治疗机会。的假设 将通过追求三个具体目标进行测试：（1）确定nosP的表型;（2）获得结构和 对NosP的功能性理解;和（3）描绘NosP下游的信号传导机制。根据目标1， P.将在存在不同量的铜绿假单胞菌的情况下定量铜绿假单胞菌生物膜、毒力和抗生素抗性。 的NO，使用野生型和NosP缺失和表达菌株。在目标2下，结构和光谱 将描述NosP的浓度，并将确定NosP是否可逆地结合NO浓度与NosP的浓度一致。 生物膜扩散在目标3下，将确定NosP是否调节某种激酶的活性以介导NosP的表达。 通过HptB/sRNA信号传导形成生物膜。PI对拟定试验具有丰富的经验。 在这些目标完成后，NosP有望被确立为调节生物膜的NO传感器 在铜绿假单胞菌中形成。这将是一个非常重要的发现，因为它将定义一个新的 信号通路和新的抗生素靶点，这是迫切需要的，特别是鉴于 增加的抗生素耐药性通常见于生物膜生物体。除了这一积极影响， 公共卫生，这项建议将对未来的研究产生重要的积极影响，因为它是一个开始， 点更深入的调查NO在生物膜调节和细菌/宿主相互作用的作用。

Bacterial biofilms are a considerable public health threat because they cause chronic and hospital-acquired infections, as well as the persistent biofouling of medical implants, but are resistant to antibiotics. Biofilm regulation by NO has been observed broadly in bacteria, thus therapeutic interventions based on NO signaling could have a significant impact on public health. Bacterial NO signaling is poorly understood, however. For example, NO regulation of biofilm dispersal in P. aeruginosa, a principal pathogen in cystic fibrosis and hospital-acquired infections, is very well documented, but the NO sensor remains unknown. 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 as a foundation for developing therapeutic strategies to disperse biofilms. The PI has shown that NO regulates biofilm formation in many bacteria, including P. aeruginosa, but P. aeruginosa lack a homolog of the H-NOX protein shown to mediate the response to NO in other species. Thus, P. aeruginosa must have an alternate NO sensor. The PI has discovered a novel family of hemoproteins named NosP. Based on strong preliminary data it is hypothesized that NosP is a NO sensor that regulates biofilm formation. The objective of the proposed work is to characterize P. aeruginosa NosP and determine the role of this protein in NO-mediated control of biofilm formation. This proposal is innovative because it forges new logical connections between NO sensing and biofilm formation, establishing a new paradigm for the role of NO in bacteria. This proposal is significant because elucidation of the basis for NO signaling in P. aeruginosa 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 determine the phenotype of nosP; (2) to gain structural and functional insights into NosP; and (3) to delineate the signaling mechanism downstream of NosP. Under aim 1, P. aeruginosa biofilm, virulence, and antibiotic resistance will be quantified in the presence of varying amounts of NO, using wild-type and NosP deletion and expression strains. Under aim 2, the structure and spectroscopy of NosP will be described and it will be determined if NosP reversibly binds NO concentrations consistent with biofilm dispersal. Under aim 3, it will be determined if NosP regulates the activity of a certain kinase to mediate biofilm formation through HptB/sRNA signaling. The PI has significant experience with the proposed assays. Upon completion of these aims, NosP is expected to be established as an NO sensor that regulates biofilm formation in P. aeruginosa. This would be a fundamentally important discovery because it will define a new signaling pathway and novel antibiotic targets, for which there is a pressing need, especially in light of the increased antibiotic resistance typically seen in biofilming organisms. In addition to this positive impact on public health, this proposal will have an important positive impact on future research, because it is the starting point for deeper investigations into the role of NO in biofilm regulation and bacterial/host interaction.