H. influenzae is a human-specific bacterial pathogen responsible for a variety of serious infections in children, including pneumonia, cellulitis, septic arthritis, and epiglottitis. Further, H. influenzae is the most common cause of bacterial meningitis in the United States, affecting approximately 12,000 children annually. Despite recent advances in antimicrobial therapy, the morbidity and mortality resulting from H. influenzae infection has changed little during the past decade. Two factors clearly distinguish strains of H. influenzae which cause invasive infection from relatively avirulent members of this species: the presence of polysaccharide capsule composed of polyribosyl ribitol phosphate (PRP) and the production of a bacteriocin protein termed haemocin. Whereas the PRP capsule has been extensively examined as a virulence factor, the role of haemocin has not been investigated. This proposal seeks to investigate the role of haemocin in the virulence of H. influenzae. To unambiguously define this role, the genetic basis of haemocin production will be determined, and the gene(s) encoding haemocin will be cloned. Mutagenesis of the cloned haemocin gene will be performed to construct isogeneic strains of H. influenzae differing only in their ability to produce haemocin. These isogeneic strains will then be used to determine the effect of haemocin production in virulence by using a relevant animal model of H. influenzae infection. The relative abilities of HMC+ and HMC-strains to colonize and to invade infant rats will be assessed. Another goal of this project is to purify and characterize the haemocin protein. The potential role of haemocin in contributing to virulence by mediating a direct toxic effect upon mammalian host cells will be examined by investigating the effects of haemocin production upon relevant human tissue in vitro. Should haemocin prove important in virulence, the availability of the cloned haemocin gene and its protein product will permit further investigations designed to define new strategies for the prevention of H. influenzae infection.
