Complex Role of Hemoglobin and Hemoglobin-Haptoglobin Binding Proteins in Haemophilus influenzae Virulence in the Infant Rat Model of Invasive Infection

Abstract

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.

Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis

“…We have seen similar age-related differences in virulence when comparing strain HI689 with a mutant derivative lacking the hemoglobin-haptoglobin binding proteins [14]. These proteins are essential for the utilization of hemoglobin-haptoglobin [9] and the age-related difference in virulence could be explained by hemoglobin-haptoglobin complexes becoming the predominant available heme source in 30-day old rats [14]. We have also recently reported age-related differences in virulence when comparing a type b strain with a mutant in the heme-hemopexin utilization genes (hxuCBA) [10].…”

“…It remains possible that there is an, as yet unidentified, additional heme source available in the 5-day old rat that becomes unavailable as the rat ages. We have seen similar age-related differences in virulence when comparing strain HI689 with a mutant derivative lacking the hemoglobin-haptoglobin binding proteins [14]. These proteins are essential for the utilization of hemoglobin-haptoglobin [9] and the age-related difference in virulence could be explained by hemoglobin-haptoglobin complexes becoming the predominant available heme source in 30-day old rats [14].…”

“…A rat model was used to assess bacteremia in both 5-day old and 30-day old rats as previously described [10,11,14]. Specified pathogen free timed-pregnant Sprague-Dawley rats were received five days prior to parturition.…”

Characterization of the Haemophilus influenzae tehB gene and its role in virulence

“…The impact of the mutation with respect to bacteraemia in 30-day-old rats was greater than that in 5-day-old rats. We have noted similar differential impacts on virulence in 5-day-old and 30-day-old rats in studies of several proteins related to haem acquisition (Morton et al, 2007a(Morton et al, , b, 2009Seale et al, 2006). For example a mutant lacking the periplasmic haem-binding protein HbpA exhibited wild-type levels of virulence in 5-day-old rats but was significantly attenuated compared to the wild-type strain in 30-day-old rats ).…”

“…Mutant genes with subtle effects on virulence, such as those with overlapping/redundant functions (e.g. genes involved Fe/Hm acquisition), often exhibit a significantly reduced ability to produce and sustain bacteraemia in weanling rats compared to infant rats (Seale et al, 2006;Morton et al, 2007aMorton et al, , b, 2009. For this reason we assessed the impact on virulence of the tehB deletion in both infant rats (5 days old) and weanling rats (30 days old) (Fig.…”