HEMIN RECEPTOR GENE FAMILY OF BARTONELLA QUINTANA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。目的：巴尔通氏杆菌是引起人类Oroya热、猫抓病、细菌性血管瘤病和战壕热的新发病原体。巴顿杆菌病的危及生命的并发症可能包括心内膜炎、病毒性肝炎、复发性菌血症、脑病和神经视网膜炎。Bartonella Quintana是这项研究的模型，目前正在市中心无家可归的人和艾滋病患者中重新出现。像所有巴氏杆菌一样，昆塔纳杆菌定植于循环系统，在那里它感染人类红细胞、血管内皮细胞，触发血管生成，并导致持续性菌血症。尽管有这些显著的特征，但人们对巴尔通体的分子发病机制知之甚少。由于氯化血红素是所有Bartonella物种的基本生长因子，而B·quintana对氯化血红素的需求是已知所有细菌中最大的，因此这项研究的长期目标是检查获得氯化血红素的分子基础--这一过程不仅有助于建立感染，而且有助于在节肢动物媒介和人类宿主中持续存在。为此，该提案重点分析了编码B.Quintana的主要氯化血红素受体的五个基因家族和四个同源物。特定目标1将使用RT-PCR来量化HBP的表达，以响应不同的氯化血红素浓度。我们还将使用凝胶迁移率改变分析和DNA足迹法分析潜在的铁摄取调节器(FUR)盒，并绘制HBP转录起始点。我们还将通过量化HBP在毛皮突变和过度表达的毛皮背景中的表达来验证Fur在HBP调节中的作用。在特定的目标2中，我们将分析HBP多基因家族在人类虱子载体和猕猴灵长类动物模型感染过程中的表达模式。此外，还将产生一个显性HBP基因的突变体和一个反式互补菌株，以在灵长类动物模型中测试分子Koch假设。在具体目标3中，我们将通过使用生化和遗传方法绘制功能受体结构域来确定HBP蛋白的结构和功能。此外，我们将确定HBP是否可以运输氯化血红素，并将确定这一功能所必需的结构域。这些数据将为多基因家族提供有价值的信息，该多基因家族参与了巴尔通体的生长和持久性的关键过程。此外，由于巴尔通体HBP可能是几种革兰氏阴性细菌外膜蛋白家族的成员，因此这项研究产生的数据无疑将对细菌的发病机制具有广泛的重要性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: Five Bartonella species are emerging infectious agents responsible for Oroya fever, cat-scratch disease, bacillary angiomatosis, and trench fever in humans. Life-threatening complications of bartonellosis can include endocarditis, peliosis hepatis, relapsing bacteremia, encephelopathy and neuroretinitis. Bartonella quintana, the model for this study, is currently re-emerging in inner-city homeless people and in patients suffering from AIDS. Like all Bartonella, B. quintana colonizes the circulatory system, where it infects human erythrocytes, vascular endothelial cells, triggers angiogenesis and causes persistent bacteremia. Despite these remarkable attributes, little is known about the molecular pathogenesis of Bartonella. Because hemin is an essential growth factor for all Bartonella species and B. quintana has the greatest hemin requirement known for any bacterium, the long range goal of this study is to examine the molecular basis for hemin acquisition-- a process that would contribute not only to establishment of infection but persistence in the arthropod vector and human host. To this end, the proposal focuses on analysis of a five-gene family encoding B. quintana's major hemin receptor and four homologues. Specific Aim 1 will quantify hbp expression in response to varying hemin concentration using RT-PCR. We will also analyze the potential ferric uptake regulator (fur) box using electrophoretic mobility shift assays and DNA footprinting, and we will map hbp transcription initiation sites. We will also verify Fur's role in hbp regulation by quantifying hbp expression in both fur mutant and over-expressed fur backgrounds. In Specific Aim 2 we will analyze the expression patterns of the hbp multigene family over the course of infection in the human louse vector and a macaque primate model. In addition, a mutant for the dominant hbp gene and a trans-complemented strain will be generated to test molecular Koch's postulates in the primate model. In Specific Aim 3, we will determine the structure and function of the Hbp proteins by mapping functional receptor domains using biochemical and genetic approaches. In addition, we will determine whether Hbp's can transport hemin and will identify domains that are necessary for this function. These data will provide valuable information on a multigene family involved in an essential process for Bartonella growth and persistence. Further, since Bartonella Hbp's are possibly members of an outer membrane protein family from several Gram-negative bacteria, data generated from this study will undoubtedly be of broad importance to bacterial pathogenesis.