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

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 铜绿假单胞菌具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，铜绿假单胞菌可以通过血红素同化（has）和 假单胞菌血红素利用 (phu) 系统。我们最近证明 OM 受体 PhuR 具有独特的 His-Tyr 配位，这是高亲和力血红素采集系统中的一个新兴主题。 13C-血红素同位素 标记研究结合细菌遗传学表明 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。此外， 我们已经证明血红素代谢物胆绿素 IXβ 是血红素传感系统 (has) 的反馈调节剂， 以及几种毒力机制，包括绿脓素和 Zn/Ni-pseudopaline 摄取系统，类型 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取的调控和分子机制。具体来说，我们将 阐明控制 has 系统表达的血红素依赖性调节元件 转录和翻译融合研究。有针对性的转录和转录后研究将 通过转录组和蛋白质组分析进行全局分析。我们将进一步定义 bis-His HasR 和 His-Tyr 协调的 PhuR 的底物特异性及其各自的贡献 血红素获取和调节。 Has 和 Phu 系统对血红素获取和毒力的贡献 将在小鼠急性和慢性肺部感染模型中对宿主内进行测试。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。 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 PAO1 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 宿主-病原体相互作用。

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.