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

• 批准号: 9916715
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916715
• 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; Image; Immune; Immune response; In Vitro; Infection; Innate Immune Response; Iron; Isomerism; Isotope Labeling; Isotopes; Knock-out; Label; Laboratories; Lead; Lung infections; Mass Spectrum Analysis; 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; Respiratory physiology; 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; multidrug-resistant Pseudomonas aeruginosa; mutant; new therapeutic target; novel; pathogen; pathogenic bacteria; promoter; protein expression; pyochelin; pyoverdin; receptor; resistant strain; sensor; trafficking; trait; transcriptome sequencing; transcriptomics; uptake

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.

致病菌需要铁来维持其生存和毒性。条件致病菌假单胞菌 铜绿假单胞菌具有多种获得铁的机制，包括铁和亚铁的摄取 系统.然而，在宿主内，铜绿假单胞菌可以通过血红素同化（has）和 假单胞菌血红素利用系统。我们最近已经证明OM受体BXR具有独特的 His-Tyr配位，这是高亲和力血红素获取系统中的新兴基序。13 C-血红素同位素 结合细菌遗传学的标记研究表明，BXR受体是高容量摄取的 受体，其中HasR受体主要作为血红素利用的传感器和调节器。此外，委员会认为， 我们已经证明血红素代谢物胆绿素IXβ是血红素感受系统（HAS）的反馈调节剂， 以及几种毒力机制，包括绿脓菌螯铁蛋白和锌/镍-假碱摄取系统， III分泌系统（ExoS和ExoT）和细胞外蛋白酶（LasB）。该提案的目标是 了解铜绿假单胞菌血红素获得的调控和分子机制。具体来说，我们将 阐明血红素依赖性调控元件控制表达的has系统， 转录和翻译融合研究。将进行靶向转录和转录后研究 通过转录组学和蛋白质组学分析进行全面分析。我们将进一步定义 bis-His HasR和His-Tyr配位的HisR的底物特异性及其各自对 血红素的获得和调节。Has和Phu系统对血红素获得和毒性的贡献 将在小鼠急性和慢性肺部感染模型中测试宿主内的作用。此外，双RNA-seq将 以同时测定铜绿假单胞菌和鼠宿主对感染的反应。MALDI- MSI将与定量LC-MS方法结合使用，以确定血红素的空间分布 PAO 1和血红素利用突变体中的代谢物（BVIX异构体）和宿主-病原体生物标志物。完成 这些研究将为铜绿假单胞菌适应血红素利用提供分子基础， 寄主-病原体相互作用