Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, USA.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, USA.
J Biol Chem. 2021 Jan-Jun;296:100275. doi: 10.1016/j.jbc.2021.100275. Epub 2021 Jan 9.
Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.
铜绿假单胞菌是一种机会致病菌，其生存和毒力都需要铁。铜绿假单胞菌可以通过非冗余血红素摄取系统和假单胞菌血红素摄取（Phu）系统从血红素中获取铁。血红素通过血红素摄取系统或 Phu 系统运输，由细胞质蛋白 PhuS 隔离。此外，PhuS 已被证明可以将血红素特异性地转运至铁调节血红素加氧酶 HemO。由于志贺氏菌的 PhuS 同源物 ShuS 被观察到作为其血红素状态的功能与 DNA 结合，因此我们试图进一步确定 PhuS 是否除了在通过 HemO 调节血红素通量方面的作用外，还作为一种 DNA 结合蛋白发挥作用。在这里，通过染色质免疫沉淀-PCR、EMSA 和荧光各向异性的组合，我们表明 apo-PhuS 但不是 holo-PhuS 结合串联铁反应性 sRNA prrF1、F2 的上游。先前的研究表明，PrrF sRNA 在铁饥饿期间对必需蛋白进行铁节约是必需的。此外，在某些条件下，PrrF1 终止子的血红素依赖性通读会产生更长的 PrrH 转录本。对铜绿假单胞菌 WT 和 ΔphuS 菌株的定量 PCR 分析表明，PhuS 的缺失消除了 PrrF 和 PrrH 水平的血红素依赖性调节。总之，我们的数据表明，PhuS 除了在细胞外血红素代谢中的作用外，还通过调节 PrrF 和 PrrH 水平来响应血红素，作为转录调节剂发挥作用。PhuS 的这种双重功能对于将细胞外血红素利用整合到 PrrF/PrrH sRNA 调控网络中至关重要，这对于铜绿假单胞菌在宿主内的适应和毒力至关重要。
