Hemin Receptor Gene Family of Bartonella quintana

描述（由申请人提供）：五种巴尔通体是导致人类奥罗亚热、猫抓病、细菌性血管瘤病和壕沟热的新出现的传染性病原体。危及生命的巴通体病并发症包括心内膜炎、肝纤维化、复发性菌血症、脑病和神经视网膜炎。这项研究的模型——金塔纳巴尔通体，目前正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，金塔纳巴尔通体在循环系统中定植，在那里它感染人的红细胞、血管内皮细胞，引发血管生成并引起持续的菌血症。尽管有这些显著的特性，但对巴尔通体的分子发病机制知之甚少。由于血红素是所有巴尔通体必不可少的生长因子，而B. quintana对血红素的需求量是已知的所有细菌中最大的，因此本研究的长期目标是研究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，本研究重点分析了一个编码金塔纳主要血红素受体和四个同源物的五基因家族。特异性Aim 1将使用RT-PCR量化hbp表达对不同血红蛋白浓度的响应。我们也将分析潜在的铁摄取调节（fur）盒使用电泳迁移转移测定和DNA足迹，我们将绘制hbp转录起始位点。我们还将通过量化皮毛突变和过表达皮毛背景下的hbp表达来验证皮毛在hbp调控中的作用。在特异性目标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，将产生显性hbp基因的突变体和反互补菌株，以在灵长类动物模型中测试分子Koch的假设。在特异性目标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是否可以运输血红蛋白，并将确定该功能所必需的结构域。这些数据将为涉及巴尔通体生长和持续的重要过程的多基因家族提供有价值的信息。此外，由于Hbp巴尔通体可能是几种革兰氏阴性菌外膜蛋白家族的成员，因此本研究产生的数据无疑对细菌的发病机制具有广泛的重要性。

DESCRIPTION (provided by applicant): 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.