The non-redundant role of the Has and Phu heme acquisition systems of Pseudomonas aeruginosa in iron homeostasis

铜绿假单胞菌 Has 和 Phu 血红素获取系统在铁稳态中的非冗余作用

• 批准号: 9469309
• 负责人: Alecia Tennessee Dent
• 金额: $ 2.92万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9469309
• 关键词: Acinetobacter; Acinetobacter baumannii; Affinity; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Assimilations; Bacteria; Biochemical; Biophysics; Cell surface; Centers for Disease Control and Prevention (U.S.); Chronic; Clinical; Complement; Coupled; Development; Enterobacter; Enterococcus faecium; Future; Genes; Genetic Transcription; Goals; Heme; Heme Iron; Homeostasis; Human body; Immune; In Vitro; Infection; Iron; Isotope Labeling; Klebsiella pneumoniae; Knock-out; Laboratories; Ligation; Lung; Membrane; Methods; Molecular; Multi-Drug Resistance; Mutation; Neisseria gonorrhoeae; Nutrient; Operon; Pathway interactions; Patients; Production; Property; Proteins; Pseudomonas; Pseudomonas aeruginosa; Receptor Gene; Regulation; Reporting; Resistance; Role; Siderophores; Sigma Factor; Signal Transduction; Site-Directed Mutagenesis; Source; Staphylococcus aureus; System; Techniques; Therapeutic; Time; United States; Virulence; antimicrobial; bacterial genetics; base; cystic fibrosis patients; experimental study; extracellular; heme receptor; in vivo; metabolomics; mortality; novel; pathogen; pathogenic bacteria; promoter; pyochelin; pyoverdin; receptor; resistant strain; sensor; transcriptome; uptake

Project Summary Pathogenic bacteria require iron for their survival and virulence. The opportunistic pathogen Pseudomonas aeruginosa has many mechanisms by which it can acquire iron, including ferric and ferrous iron uptake systems. However, within the host P. aeruginosa adapts to utilize heme as an iron source via the heme assimilation (has) and Pseudomonas heme utilization (phu) systems. The has operon encodes an extracellular hemophore, HasAp that scavenges heme and transfers it to the outer membrane (OM) receptor, HasR which acts as the cell surface signaling (CSS) system. The hemophore and receptor genes are downstream of an Extra Cytoplasmic Function (ECF) sigma and anti-sigma factor (HasI and S, respectively) associated with signal transduction and regulation of heme uptake. We have recently shown that in contrast to the bis-His coordination of HasR, the OM receptor PhuR has a unique His-Tyr coordination, an emerging motif in high affinity heme acquisition systems. 13C-heme isotopic labeling studies in combination with bacterial genetics suggested the PhuR receptor is the high capacity uptake receptor, with the HasR receptor acts primarily as a sensor and regulator of heme utilization. The goal of my proposal will be to determine the contributions of the Has and Phu systems to heme sensing and transport through 1) identifying the downstream targets of the HasAp-HasR cell surface signaling system (CSS) and its extra-cytoplasmic function (ECF) sigma factor HasI; and 2) determine the contributions of the HasR and PhuR receptors to heme uptake and utilization. We will utilize a combination of in vitro biochemical and spectroscopic techniques to characterize the molecular mechanism of heme regulation and uptake in combination with in vivo techniques including bacterial genetics and 13C-heme isotopic labeling coupled to LC-MS/MS to determine heme uptake in knockout and complemented strains. At the conclusion of this project I will have determined the regulatory cascade of the Has heme sensing system and the contributions of the non-redundant Has and Phu systems in heme utilization by P. aeruginosa. This study will provide a platform for the identification of novel antimicrobial therapies.

项目概要 致病细菌需要铁来维持其生存和毒力。机会致病菌假单胞菌 铜绿假单胞菌有多种获取铁的机制，包括三价铁和二价铁的吸收 系统。然而，在宿主体内，铜绿假单胞菌通过血红素适应利用血红素作为铁源。 同化（has）和假单胞菌血红素利用（phu）系统。 has操纵子编码细胞外 血红素 HasAp 清除血红素并将其转移到外膜 (OM) 受体 HasR 充当细胞表面信号传导（CSS）系统。血细胞和受体基因位于下游 细胞质外功能 (ECF) sigma 和抗 sigma 因子（分别为 HasI 和 S）与 血红素摄取的信号转导和调节。我们最近表明，与双组氨酸相比 HasR 的协调，OM 受体 PhuR 具有独特的 His-Tyr 协调，这是高水平中的新兴基序 亲和血红素采集系统。 13C-血红素同位素标记与细菌遗传学相结合的研究 表明 PhuR 受体是高容量摄取受体，HasR 受体主要充当 血红素利用的传感器和调节器。我的提案的目标是确定 Has 和 Phu 系统通过 1) 识别血红素的下游目标来进行血红素传感和运输 HasAp-HasR细胞表面信号系统（CSS）及其胞质外功能（ECF）西格玛​​因子HasI； 2) 确定 HasR 和 PhuR 受体对血红素摄取和利用的贡献。我们将 利用体外生化和光谱技术的组合来表征分子 与包括细菌遗传学在内的体内技术相结合的血红素调节和摄取机制 13C-血红素同位素标记与 LC-MS/MS 联用，以确定敲除和 补充菌株。在这个项目结束时，我将确定监管级联 Has血红素传感系统以及非冗余Has和Phu系统对血红素的贡献 铜绿假单胞菌的利用。本研究将为新型抗菌药物的鉴定提供平台 疗法。