Integration of heme acquisition and signaling in Gram-negative pathogens

项目总结 ESKAPE病原体是耐药感染的主要原因，需要寻找新的抗菌药物 战略至关重要。铁是生存和毒力所必需的微量营养素，而微生物病原体 主动地将铁从微生物中隔离出来。病原体通过多种途径克服这种铁限制。 机制，包括铁载体的合成和分泌，清除铁(Fe3+)铁， 通过FeO或类NRAMP系统吸收亚铁(Fe2+)铁，并从宿主血红素中获得铁。铁质 微生物病原体的获取和动态平衡是多因素的，依赖于复杂的 转录和转录后调控网络。我们最近展示了PHU的细胞质 血红素结合蛋白具有调节血红素通过血液的流动和转录的双重功能 铁和血红素的调节调节了sRNA的PrrF和PrrH。PrrF sRNA与互补性结合 它们的靶RNA序列导致相关基因的RNAseE和依赖于Hfq的mRNA降解 在铁的储存和氧化应激中，有氧和无氧代谢，包括含铁的蛋白质 三氯乙酸循环，以及几个毒力因子。因此，通过血液调节血红素流量是一种 血红素代谢和铁依赖的sRNA调控网络之间的关键联系 寄主内的适应性和致病力。该提案的目标是在分子水平上了解 血红素的收购被整合到这些监管网络中。具体地说，我们将；i)确定PHU 血红素转移和结合prrF1启动子所需的结构基序(PprrF1)，II)定义体内 PHU变异对PrrF/H sRNAs及其下游调控蛋白的影响以及三)确定进化 肠道病原菌间PHU同源物的保守性和功能。在完成研究后，我们会 已经确定了细胞外血红素代谢在三种铁依赖的调节网络中的作用 重要的人类病原体，提供了一个平台，在 铁稳态与毒力之间的界面。

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.