H. influenzae type b is a common childhood pathogen which causes serious invasive infections such as meningitis and pneumonia. In the United States, approximately 10,000 infants acquire memingitis due to H. influenzae each year. Approximately 500, of these infants die, and neurologic sequelae are detectable in 50% of the survivors despite antibiotic therapy. Although H. influenzae requires iron for growth, the source of iron in invasive infections is unclear. The concentration of unbound iron in serum is approximately 10-18M. Most microbial species require an iron concentration of approximately 10-6M for growth; therefore, many microbial pathogens have developed inducible mechanisms for the specific uptake of iron to compete effectively. In addition to iron, H. influenzae requires porphyrin for growth, which is usually supplied as heme. This proposal seeks to investigate the role of heme and non-heme iron acquisition in the pathogenesis of invasive infections due to H. influenzae type b. The presence of low molecular weight iron-binding compounds called siderophores will be investigated by biochemical assays for phenolates and hydroxamates and bioassays using mutants to detect siderophore usage or production of H. influenzae. The ability of iron sources present in vivo, e.g. transferrin, hemoglobin, heme-hemopexin, to sequester iron from H. influenzae will be determined in complex, defined media. The presence of new outer membrane proteins induced by iron starvation will be determined by SDS-PAGE of outer membranes of pathogenic and nonpathogenic strains of H. influenzae. The role of these new outer membrane proteins as possible receptors for iron containing compounds will be determined by in situ binding of radiolabelled iron containing compounds and binding after Western blotting. The presence of absence of plasmids will be determined by agarose gel electrophoresis of whole cell lysate. Mutants defective in iron uptake will be characterized by analysis of siderophore production and alteration in their outer membrane protein composition. The importance of iron acquisition as a virulence factor will be determined in infant rats by comparison of the quantitative bacteremia of isogenic strains and protection experiments using iron binding human serum proteins. Knowledge of the mechanisms of specific acquisition of a critical nutrient such as iron will provide the basis for defining the genetics of these pathogenic mechanisms and the role of host serum proteins in defense against invasive pathogens.
