Bartonellosis or Carrion's disease is often a biphasic illness caused by Bartonella bacilliformis, a gram-negative bacteria transmitted by the Phlebotomine sandfly in the valleys of Peru, Ecuador and Colombia. The Oroya fever phase of the disease is a severe, highly fatal, hemolytic anemia characterized by the presence of the organism within erythrocytes and in the cytoplasm of reticuloendothelial and vascular endothelial cells. The tissue phase of the disease, verruga peruana, is characterized by the formation of wart-like lesions and nodules on the skin that vary in size from small nodules to giant tumors. The long term objective of this research is to elucidate the molecular mechanisms that are utilized by the parasite to gain entry inside host cells and to identify the nature of the receptor on host cells.
The Specific Aims of this study are: l) the isolation and characterization of invasion gene determinants from a Bartonella bacilliformis gene bank constructed in the Bluescript cloning vector. Immunoscreening of clones will be carried out using anti-B.bacilliformis immune serum. Further characterization will include restriction endonuclease mapping of the isolated gene fragment and determination of the precise coding region using standard subcloning experiments and transposon mutagenesis. Nucleotide sequencing will be carried out using the Sanger dideoxynucleotide chain termination method. 2) the expression and analysis of the invasion gene product using the coupled T7 RNA polymerase/promoter system in Bluescript and SDS-PAGE and Western blotting. 3) identification and characterization of the Bartonella receptor on host cells using affinity column chromatography and 4) assessment of the role of signal transduction pathways in Bartonella entry of host cells using inhibitors of tyrosine protein kinase activity and inhibitors of other signal transduction pathway intermediates.
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