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

Summary Haemophilus 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, HxuAdm, 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 haem–haemopexin 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 106 M−1). HxuA thus appears to ‘release’ haem from haem–haemopexin complexes and to prevent haem sequestering by haemopexin.