A gene cluster involved in the utilization of both free heme and heme:hemopexin by Haemophilus influenzae type b

Abstract

The utilization of heme bound to the serum glycoprotein hemopexin by Haemophilus influenzae type b (Hib) strain DL42 requires the presence of the 100-kDa heme:hemopexin-binding protein encoded by the hxuA gene (M. S. Hanson, S. E. Pelzel, J. Latimer, U. Muller-Eberhard, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). Nucleotide sequence analysis of a 5-kb region immediately upstream from the hxuA gene revealed the presence of two genes, designated hxuC and hxuB, which encoded outer membrane proteins. The 78-kDa HxuC protein had similarity to TonB-dependent outer membrane proteins of other organisms, whereas the 60-kDa HxuB molecule most closely resembled the ShlB protein of Serratia marcescens. A set of three isogenic Hib mutants with cat cartridges inserted individually into their hxuA, hxuB, and hxuC genes was constructed. None of these mutants could utilize heme:hemopexin. The hxuC mutant was also unable to utilize low levels of free heme, whereas both the hxuA and hxuB mutants could utilize free heme. When the wild-type hxuC gene was present in trans, the hxuC mutant regained its ability to utilize low levels of free heme but still could not utilize heme:hemopexin. The hxuA mutant could utilize heme:hemopexin when a functional hxuA gene from a nontypeable H. influenzae strain was present in trans. Complementation analysis using this cloned nontypeable H. influenzae hxuA gene also indicated that the HxuB protein likely functions in the release of soluble HxuA from the Hib cell. These studies indicate that at least two and possibly three gene products are required for utilization of heme bound to hemopexin by Hib strain DL42.

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

“…HxuB is believed to be involved in the secretion of the soluble HxuA molecule from the bacterial cell (Cope et al, 1995). 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).…”

“…HxuC possesses the amino acid sequences considered characteristic of TonB-dependent proteins that are typically involved in the binding and transport of certain lowmolecular-mass ligands (Postle, 1990;Cope et al, 1995). HxuB is believed to be involved in the secretion of the soluble HxuA molecule from the bacterial cell (Cope et al, 1995). Mutations in hxuA and/or hxuB abolish the ability to utilize the haem-haemopexin complex (Cope et al, 1995).…”

“…The HxuA-haemhaemopexin complex is presumably bound by the bacterial cell and the haem is internalized; the outer-membrane moiety mediating the binding of HxuA has not been identified although the receptor may be HxuC (Cope et al, 1998). HxuC possesses the amino acid sequences considered characteristic of TonB-dependent proteins that are typically involved in the binding and transport of certain lowmolecular-mass ligands (Postle, 1990;Cope et al, 1995). HxuB is believed to be involved in the secretion of the soluble HxuA molecule from the bacterial cell (Cope et al, 1995).…”

“…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 .…”

“…et al, 1999, 2004b). Utilization of the haem-haemopexin complex is mediated by the products of the hxuCBA gene cluster (Hanson et al, 1992;Cope et al, 1995). HxuA is a 100 kDa secreted protein that binds the haem-haemopexin complex (Cope et al, 1994(Cope et al, , 1998.…”