Brazilian purpuric fever (BPF) is a fatal disease caused by a unique invasive clone of Haemophilus influenzae biogroup aegyptius (H. aegyptius), a bacterial species that is normally non-invasive and causes mild cases of conjunctivitis. The unusual invasive attributes of the BPF clone of H. aegyptius make this pathogen an ideal model to study the emergence of an invasive derivative, which expresses new virulence determinants, from an old bacterial pathogen. Furthermore, the availability of a tissue culture model using the natural host-target cells and the existence interactions, bacterial gene transfer, and the regulation of the expression of bacterial virulence genes. In addition, little is known about the molecular and genetic nature of the BPF virulence factors and the pathogenesis of the purpura fulminans caused by this pathogen. Thus, the long-term goal of this research project is the elucidation of the genetic and molecular mechanisms involved in the invasion and destruction of the endothelial host-cells by the BPF clone. In this proposal, we address this overall goal through several approaches, combining classical methods used in bacterial genetics with techniques designed to isolate unique genes present in virulent strains and examine differential gene expression. The first Specific Aim involves the isolation and characterization of unique BPF genes, some of which may be involved in virulence. This will be achieved by constructing a gene library using the innovative subtraction genomic hybridization technique. The second Specific Aim is focused on the expression analysis of the unique BPF genes at the transcriptional and translational levels using northern and western blotting together with reverse transcriptase- polymerase chain reaction. The third Specific Aim examines the role of unique BPF genes in the invasion and destruction of human endothelial cells using isogenic mutants. These mutants, which will be generated by site-directed insertion mutagenesis and allelic exchange, will be tested in the HMEC-1 tissue culture model that mimics the vascular destruction produced during the infection process caused by the invasive strains of H. aegyptius. These proposed studies address an important and largely unexplored aspect of the pathogenesis of the vascular destruction caused by the BPF clone of H. aegyptius. Furthermore, these studies will lead to a better comprehension of the nature of other vascular destructive infectious diseases.
