HEMIN RECEPTOR GENE FAMILY OF BARTONELLA QUINTANA

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目的：五种巴尔通体是引起人类奥罗亚热、猫抓病、杆菌性血管瘤病和战壕热的新出现的传染性病原体。巴氏杆菌病的致命并发症包括心内膜炎、肝炎、复发性菌血症、脑病和神经视网膜炎。本研究的模型巴尔通体目前在市中心无家可归者和艾滋病患者中重新出现。像所有巴尔通体一样，B。喹他那定殖于循环系统，感染人体红细胞和血管内皮细胞，引发血管生成，并导致持续性菌血症。尽管有这些显著的属性，但对巴尔通体的分子发病机制知之甚少。因为氯化血红素是所有巴尔通体属种和B的必需生长因子。quintana对氯化血红素的需求是所有细菌中最大的，本研究的长期目标是检查氯化血红素获取的分子基础--这一过程不仅有助于建立感染，而且有助于在节肢动物载体和人类宿主中持续存在。为此，该提案侧重于分析一个编码B的五基因家族。quintana的主要氯化血红素受体和四个同系物。特异性目的1将使用RT-PCR定量hbp表达对不同氯化血红素浓度的响应。我们还将使用电泳迁移率变动分析和DNA足迹分析潜在的铁吸收调节（毛皮）盒，我们将映射hbp转录起始位点。我们还将通过定量hbp在毛皮突变体和过度表达的毛皮背景中的表达来验证毛皮在hbp调节中的作用。在具体目标2中，将分析hbp多基因家族在人虱载体和猕猴灵长类动物模型中感染过程中的表达模式。此外，将产生显性hbp基因的突变形式和反式互补菌株，以在灵长类动物模型中测试分子科赫假设。在具体目标3中，我们将通过使用生物化学和遗传学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，我们将确定是否Hbp的可以运输氯化血红素，并确定这一功能所必需的域。这些数据将提供有关巴尔通体生长和持久性的重要过程中所涉及的多基因家族的有价值的信息。此外，由于巴尔通体Hbp可能是来自几种革兰氏阴性菌的外膜蛋白家族的成员，因此本研究产生的数据无疑对细菌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 the expression patterns of the hbp multigene family over the course of infection in the human louse vector and a macaque primate model will be analyzed. In addition, a mutant form of 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.