The long-term objective of this research proposal is the molecular and genetic dissection of the iron acquisition systems in Haemophilus influenzae biogroup aegyptius (H. aegyptius) strains isolated from cases of Brazilian purpuric fever (BPF). BPF is a fulminant septicemic disease in children caused by a single clone of H. aegyptius, referred to as the BPF clone. Although substantial progress has been made since the description of the first case of BPF in 1985, the factor(s) responsible for the enhanced virulence of the BPF clone remains unknown. Gene products and regulatory mechanisms that are essential for bacterial survival in the host are considered substantial components of the bacterial virulence repertoire. This is an important aspect in the biology of the H. aegyptius BPF clone as it must invade the conjunctiva and/or nasopharyngeal mucosa, survive and multiply in the blood to produce the typical histopathologic lesions of BPF. During this process, the bacteria must acquire essential nutrients such as iron, a metal that, although abundant in the host, is not freely available for microbial growth. Mechanisms whereby a pathogenic bacterium can utilize this otherwise unavailable iron are important in bacterial virulence. Currently, the molecular mechanisms of iron utilization by H. aegyptius are unknown and preliminary data from several laboratories working on H. influenzae suggest that the iron-uptake systems in this bacterium are complex. Thus, the examination of iron acquisition and the regulation of its expression are significant areas for pathogenesis-control and prevention-related studies on this fulminant disease. Furthermore, the knowledge gained during this study will increase our understanding of the pathogenesis of other bacterial invasive diseases caused by H. influenza strains different from the classical type b isolates. To achieve these goals, the research plan includes: (1) genetic and molecular characterization of the transferrin-binding proteins (TBP1 and TBP2). Generation of TBP1 and/or TBP2 isogenic mutants will permit assessment of the role of these proteins in iron acquisition; (2) identification and analysis of the components associated with the transfer of iron from the surface receptor to the cytosol. The existence of Fbp- and TonB-like proteins will be evaluated by molecular cloning and DNA sequencing, mutagenesis, and complementation assays; (3) characterization of the regulation of the expression of iron- repressed proteins. The presence of a repressor homologous to the E. coli Fur protein will be investigated by cloning and mutagenesis analysis. In addition, a novel genetic approach (Fur titration assay) will be used to identify new Fur-regulated genes in H. aegyptius.
