Integration of heme acquisition and signaling in Gram-negative pathogens

项目摘要 ESKAPE病原体是耐药性感染的主要原因，需要确定新的抗菌药物 战略至关重要。铁是微生物病原体生存和毒力所必需的微量营养素， 积极地将铁与微生物隔离。病原体通过多种途径克服这种铁限制 的机制，包括合成和分泌的铁载体，使铁（Fe 3+）铁， 通过FeO或NRAMP样系统摄取亚铁（Fe 2+）铁，以及从宿主血红素中获取铁。铁 微生物病原体的获得和体内平衡是多因素的， 转录和转录后调控网络。我们最近发现， 血红素结合蛋白具有双重功能，在调节血红素通过HemO的通量中，以及在转录调控中， 铁和血红素的调节调节sRNA的PrrF和PrrH。PrrF sRNA结合互补的 它们的靶RNA序列引起相关基因的RNAseE和Hfq依赖性mRNA降解 在铁储存和氧化应激，有氧和无氧代谢，包括铁的含蛋白质， TCA循环，以及几个毒力因子。因此，通过HemO调节血红素通量是一个重要的因素。 血红素代谢和铁依赖性sRNA调控网络之间的关键联系， 适应性和宿主内的毒性。该提案的目标是在分子水平上了解 血红素获取被整合到这些调节网络中。具体而言，我们将：i）确定 血红素转移和结合prrF 1启动子（PprrF 1）所需的结构基序，ii）定义了体内 PrrF/H sRNA和下游调节子的影响。（三）确定进化 在肠道病原体中的C37 S同源物的保守性和功能。研究完成后，我们会 已经确定了细胞外血红素代谢在三种铁依赖性调节网络中的作用， 重要的人类病原体，提供了一个平台，为确定抗菌策略， 铁稳态和毒力之间的界面。

PROJECT SUMMARY ESKAPE pathogens are a leading cause of drug resistant infections and the need to identify new antibacterial strategies is critical. Iron is an essential micronutrient for survival and virulence that microbial pathogens which actively sequesters iron away from microorganisms. Pathogens overcome this iron limitation through a variety of mechanisms, including the synthesis and secretion of siderophores that scavenge ferric (Fe3+) iron, the uptake of ferrous (Fe2+) iron via Feo or NRAMP-like systems, and acquisition of iron from host heme. iron acquisition and homeostasis by microbial pathogens is multifactorial and dependent on sophisticated transcriptional and post-transcriptional regulatory networks. We have recently shown the PhuS cytoplasmic heme binding protein has a dual function in regulating heme flux through HemO, and in the transcriptional regulation of the iron and heme regulated sRNA’s PrrF and PrrH. The PrrF sRNAs bind to complementary sequences of their target RNAs causing the RNAseE and Hfq-dependent mRNA degradation of genes involved in iron-storage and oxidative stress, aerobic and anaerobic metabolism, including iron containing proteins of the TCA cycle, as well as several virulence factors. Therefore, regulation of the heme flux through HemO is a critical link between heme metabolism and the iron-dependent sRNA regulatory network required for adaptation and virulence within the host. The goal of the proposal is to understand at a molecular level how heme acquisition is integrated into these regulatory networks. Specifically, we will; i) determine the PhuS structural motifs required for heme transfer and binding to the prrF1 promoter (PprrF1), ii) define the in vivo effects of PhuS variants on PrrF/H sRNAs and the downstream regulon. and iii) determine evolutionary conservation and function of PhuS homologs across enteric pathogens. On completion of the studies we will have determined the role of extracellular heme metabolism in the iron-dependent regulatory networks of three significant human pathogens, providing a platform for the identification of antibacterial strategies at the interface between of iron homeostasis and virulence.