Complex role of hemoglobin and hemoglobin-haptoglobin binding proteins in Haemophilus influenzae virulence in the infant rat model of invasive infection

Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.