Mechanistic characterization and regulation of the non-redundant phu and has heme uptake systems of Pseudomonas aeruginosa

铜绿假单胞菌非冗余phu和血红素摄取系统的机制表征和调控

基本信息

批准号：
10383767
负责人：
Angela Wilks
金额：
$ 38.33万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10383767
关键词：
Acute Affinity Alleles Anabolism Anti-Bacterial Agents Antibiotics Assimilations Biliverdine Biochemical Biological Markers Chronic Clinical Data Development Drug resistance Feedback Future Genetic Genetic Transcription Goals Growth Heme Hemoglobin Immune Immune response In Vitro Infection Innate Immune Response Iron Isomerism Isotope Labeling Isotopes Knock-out Label Laboratories Lead Lung infections Membrane Messenger RNA Metabolism Methods Modeling Molecular Multi-Drug Resistance Mus Mutation Nosocomial Infections Operon Pathogenesis Patients Peptide Hydrolases Periodicity Post-Transcriptional Regulation Proteins Proteomics Pseudomonas Pseudomonas aeruginosa Regulation Regulatory Element Role Siderophores Signal Transduction Site-Directed Mutagenesis Source Spatial Distribution Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Substrate Specificity System Techniques Testing Time Transcriptional Regulation Type III Secretion System Pathway Virulence Virulence Factors World Health Organization acute infection bacterial genetics base chronic infection cystic fibrosis patients defined contribution experimental study extracellular in vivo innovation insight mass spectrometric imaging multidrug-resistant Pseudomonas aeruginosa mutant new therapeutic target novel opportunistic pathogen pathogen pathogenic bacteria promoter protein expression pulmonary function decline pyochelin pyoverdin receptor resistant strain sensor trafficking trait transcriptome sequencing transcriptomics uptake

项目摘要

Pathogenic bacteria require iron for their survival and virulence. The opportunistic pathogen Pseudomonas aeruginosa has multiple mechanisms by which it can acquire iron, including ferric and ferrous iron uptake systems. However, within the host P. aeruginosa can adapt to utilize heme via the heme assimilation (has) and Pseudomonas heme utilization (phu) systems. We have recently shown the OM receptor PhuR has a unique His-Tyr coordination, which is an emerging motif in high affinity heme acquisition systems. 13C-heme isotopic labeling studies combined with bacterial genetics suggest the PhuR receptor is the high capacity uptake receptor, with the HasR receptor acting primarily as a sensor and regulator of heme utilization. Furthermore, we have shown the heme metabolite biliverdin IXβ is a feedback regulator of the heme sensing system (has), as well as several virulence mechanisms including the pyochelin and Zn/Ni-pseudopaline uptake system, Type III secretion systems (ExoS and ExoT), and extracellular proteases (LasB). The goal of the proposal is to understand the regulation and molecular mechanism of heme acquisition in P. aeruginosa. Specifically, we will elucidate the heme-dependent regulatory elements controlling expression of the has system through transcriptional and translational fusion studies. Targeted transcriptional and post-transcriptional studies will be complimented by global analysis through transcriptomic and and proteomic analyses. We will further define the substrate specificity of the bis-His HasR and His-Tyr coordinated PhuR and their respective contributions to heme acquisition and regulation. Contributions of the Has and Phu systems to heme acquisition and virulence within the host will be tested in murine acute and chronic lung infection models. In addition, dual RNA-seq will be performed to simultaneously determine the P. aeruginosa and murine host response to infection. MALDI- MSI will be used in combination with quantitative LC-MS methods to determine the spatial distribution heme metabolites (BVIX isomers) and host-pathogen biomarkers in PAO1 and heme utilization mutants. Completion of the studies will provide a molecular basis for P. aeruginosa adaption to heme utilization in the context of the host-pathogen interaction.

致病细菌需要铁才能生存和病毒。机会性病原体假单胞菌铜绿菌具有多种机制，可以通过这些机制获取铁，包括铁和铁铁吸收系统。但是，在宿主的铜绿假单胞菌中可以通过血红素同化（HAS）和假单胞菌血红素利用率（PHU）系统。我们最近显示了OM接收器Phur具有独特的 HIS-TYR协调，这是高亲和力血红素获取系统中新兴的主题。 13C血红素同位素与细菌遗传学结合的标记研究表明，Phur受体是高容量的吸收受体，HOSR受体主要起作用是血红素利用的传感器和调节剂。此外，我们已经表明，血红素代谢产物IXβ是血红素传感系统（HAS），）的反馈调节剂以及几种病毒机制在内 III分泌系统（Exos和Exot）和细胞外蛋白酶（LASB）。该提议的目的是了解铜绿假单胞菌中血红素获取的调节和分子机制。具体来说，我们会的阐明控制Heme依赖性调节元件，以控制其通过转录和翻译融合研究。针对性的转录和转录后研究将是通过转录组和蛋白质组学分析的全球分析表示赞赏。我们将进一步定义双HIS HASR的底物特异性和His-Tyr协调的Phur及其对各自的贡献血红素的获取和调节。 HAS和PHU系统对血红素的贡献和病毒的贡献在鼠急性和慢性肺部感染模型中将测试宿主内部。此外，双RNA-Seq将进行同时确定铜绿假单胞菌和鼠宿主对感染的反应。马尔代 MSI将与定量LC-MS方法结合使用，以确定空间分布血红素 PAO1和血红素使用突变体中的代谢物（BVIX异构体）和宿主 - 病原生物标志物。完成研究将为铜绿假单胞菌适应血红素利用的分子基础宿主 - 病原体相互作用。