Heme utilization and homeostasis in Pseudomonas aeruginosa

描述(申请人提供)：病原菌需要铁才能生存和引起感染。许多细菌病原体已经进化出复杂的系统，利用血红素作为铁的主要来源。机会致病菌铜绿假单胞菌是医院感染的一种越来越常见的原因，与此类感染相关的高发病率。除了对许多抗生素的天然耐药性外，这类感染的惊人增长突出表明有必要采取替代的抗菌策略。在以前的生物化学中 和生物物理研究，我们已经表征了细胞内的血红素运输蛋白PHU和铁调节的血红素加氧酶HEMO，并进一步表明它们对血红素的利用和毒力至关重要。该提案的具体目的是进一步阐明细胞外血红素的利用和调节机制，因为它与毒力有关。这将是 通过：i)阐明细胞内的血红素转运途径，并确定胆绿素IX-β和-β异构体在通过遗传、生化和代谢组学方法对血红素吸收的细胞外反馈调节中的作用。Ii)通过定点突变、生化和生物物理研究相结合的方法，确定血红素从PHU向HEMO转移的机制；以及III)确定调节的小RNA PrrH在血红素吸收和利用中的作用。这些研究将为细菌病原体中的血红素和铁的动态平衡提供新的见解。通过这项提议，我们将确定新的血红素代谢和调控途径作为新的抗微生物靶点，并促进目前对血红素转移机制的理解。

DESCRIPTION (provided by applicant): Pathogenic bacteria require iron for their survival and ability to cause infection. Many bacterial pathogens have evolved sophisticated systems to utilize heme as a primary source of iron. The opportunistic pathogen P. aeruginosa is an increasingly common cause of nosocomial infections with high morbidity associated with such infections. In addition to their natural resistance to many antibiotics the alarming increase in such infections highlights the need for alternate antimicrobial strategies. In previous biochemical and biophysical studies we have characterized the intracellular heme trafficking protein PhuS and the iron-regulated heme oxygenase HemO and have further shown they are critical for heme utilization and virulence. The specific aims of the proposal are to further elucidate the mechanism of extracellular heme utilization and regulation as it relates to virulence. This will be achieved through; i) Elucidating the intracellular heme trafficking pathway and defining the role of biliverdin IX-delta and -beta isomers in the extra-cellular feedback regulation of heme uptake through genetic, biochemical, and metabolomics approaches. ii) Defining the mechanism of heme transfer from PhuS to HemO through a combination of site-directed mutagenesis, biochemical and biophysical studies and iii) Determining the role of the regulatory small RNA PrrH in heme uptake and utilization. These studies will provide new insights into heme and iron homeostasis in bacterial pathogens. We will through this proposal identify new heme metabolic and regulatory pathways as novel antimicrobial targets and advance the current understanding of heme transfer mechanisms.