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.
