Involvement of HxuC Outer Membrane Protein in Utilization of Hemoglobin by Haemophilus influenzae

Abstract

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobinhaptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.

The haem–haemopexin utilization gene cluster (hxuCBA) as a virulence factor of Haemophilus influenzae

“…It has also been implicated in the acquisition of haem from haemoglobin in the absence of the Hgps (Cope et al, 2001). In view of the role of HxuC in the acquisition of haem from these various sources and the localization of this protein to the outer membrane, it is likely that HxuC represents an essential component of a common high-affinity haem acquisition pathway (Cope et al, 1995;Hanson et al, 1992;Morton & Stull, 2004 (Koskelo & Muller-Eberhard, 1977)].…”

“…Mutations in hxuA and/or hxuB abolish the ability to utilize the haem-haemopexin complex (Cope et al, 1995). HxuC is required for the utilization of both the haemhaemopexin complex and low levels of free haem (Cope et al, 1995); HxuC has also been implicated in the residual utilization of haemoglobin seen in an hgp knockout background (Cope et al, 2001). Transcripts of the gene encoding HxuC can be detected in the human middle ear during acute otitis media (Whitby et al, 1997), and transcription of all three genes in the hxuCBA gene cluster is upregulated under conditions of haem starvation as would be encountered in the human host .…”

The OxyR Regulon in Nontypeable Haemophilus influenzae

“…All exhibit H 2 O 2 -induced, OxyR-independent upregulation of expression. The hxu system in H. influenzae type b encodes a heme utilization system that captures heme sequestered in a complex with hemopexin (14)(15)(16). The TonB-ExbBD system has a vital role in transducing the energy of the proton motor force to generate conformational changes in TonB-dependent outer membrane transporters (68).…”

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

“…If haptoglobin levels were low compared to hemoglobin concentrations in infant rat plasma, then free hemoglobin could potentially be available as a heme source. While hemoglobin-haptoglobin complexes cannot be utilized as a heme source for aerobic growth by the ⌬hgp mutant strain HI1717, this mutant and the wild-type strain can utilize free hemoglobin for growth in vitro, since this hemoprotein is utilized by both hgp-dependent and hgp-independent mechanisms (6,39,40,43). Thus, a haptoglobin concentration insufficient to bind all or most of the extracellular hemoglobin might permit the Hgp-deficient mutant to proliferate in vivo.…”

“…The hxuCBA gene cluster has been implicated in binding and utilization of low concentrations of free heme, hemehemopexin complexes, and hemoglobin but does not mediate utilization of hemoglobin-haptoglobin as a heme source (6,7,9,40). To further verify the presence of plasma heme sources in addition to hemoglobin-haptoglobin, we constructed two additional isogenic mutants of strain HI689, an hxuC mutant (strain HI1787) and a quadruple mutant in which the hgp genes and the hxuC gene were deleted (strain HI1810).…”