Haem release from haemopexin by HxuA allows Haemophilus influenzae to escape host nutritional immunity

Abstract

SummaryHaemophilus influenzae is an obligate human commensal/pathogen. This haem auxotroph must acquire haem from its host to sustain aerobic growth. Haem-haemopexin complexes are one of the potential sources of haem for this microorganism. Haemopexin is a glycoprotein that binds haem with high affinity (subpicomolar Kd) and involved in haem recycling. HxuA, a cell surface protein, is the key to haem acquisition from haemopexin. In this study, we reconstituted a functional Hxu system from H. influenzae in Escherichia coli K-12 that mediated active haem transport across the outer membrane from haem-haemopexin, in the presence of the inner membrane energy-transducing TonB-ExbB-ExbD complex from H. influenzae. A secreted variant of HxuA, HxuA dm, was produced in E. coli. HxuAdm functionally complemented an hxuA mutant of H. influenzae for haem-haemopexin acquisition. HxuAdm interacted with haemopexin and haem-haemopexin, with which it formed high-affinity, stoichiometric complexes. Following the interaction between haemhaemopexin and HxuAdm, haem was no longer bound to its initial high-affinity site and became accessible to its cognate haem receptor, HxuC. HxuAdm and the HxuAdm-haemopexin complex do not appear to bind haem at detectable levels (affinities below 10 6 M -1 ). HxuA thus appears to 'release' haem from haemhaemopexin complexes and to prevent haem sequestering by haemopexin.

Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae

“…H. influenzae is unable to synthesize heme but can utilize ferrochelatase to catalyze the insertion of iron into protoporphyrin IX, the precursor of heme (67), or mediate heme uptake directly from the environment (68)(69)(70)(71). The role of Fur in iron homeostasis of H. influenzae has not been explicitly described.…”

Haemophilus influenzae stores and distributes hemin by using Protein E

“…In fact, the free iron concentration is only ≈10 −18 M within the human host (Bullen and Ward, 1988). However, freely available iron may occasionally arise during tissue injury, and hence free heme is released from erythrocytes upon intravascular hemolysis and inflammation caused by 4 polymorphonuclear neutrophils (Aruoma et al, 1988;Evans et al, 1994;Skaar, 2010 (Fournier et al, 2011;Jin et al, 1996;Jin et al, 1999;Khan et al, 2007;Mason et al, 2011). These proteins are located in the periplasm, but HxuA can also be secreted.…”

Protection against Heme Toxicity: Hemopexin Rules, OK?

“…170 The low free heme and iron levels in plasma and other biological fluids, due in part to HPX, Hp, and transferrin, collectively provide individual signals that stimulate the production of a variety of metal sequestering systems by human pathogens. These systems have been well characterized in both Gram-negative and Gram-positive bacteria (Fournier et al 136 and references therein). Receptors are known for heme, Hb, Hb-Hp and heme-HPX on the plasma membrane of a variety of human pathogens, including Haemophilus influenzae as well as for transferrin and lactoferrin receptors, expressed, respectively, in Neisseriaceae and Pasteurella species.…”

“…The cell-surface protein HxuA mediates heme transport from heme-HPX across the outer membrane using energy from the TonB-ExbB-ExuD complex. 136 A secreted variant of HxuA that complements an hxuA mutant of H. influenza forms a high affinity 1:1 complex with heme-HPX, which then rapidly releases heme to a cognate heme receptor, HxuC.…”

“…HasA has a specific receptor, HasR, and the extremely tight binding of heme by HPX is overcome by the hemophore. 136 The HPX-binding proteins of H. influenzae have been characterized. The cell-surface protein HxuA mediates heme transport from heme-HPX across the outer membrane using energy from the TonB-ExbB-ExuD complex.…”