A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae

Commensal bacteria serve as an important line of defense against colonisation byopportunisitic pathogens, but the underlying molecular mechanisms remain poorlyexplored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purifiedthe NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin geneusing proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-bindingprotein folds, suggesting that it evolved independently. Biochemical characterisationshows that heme can be captured in the ferrous or ferric state, and with a variety ofsmall heme-ligands bound, suggesting that hemophilin could function under a rangeof physiological conditions. Hemophilin knockout bacteria show a limited capacity toutilise free heme for growth. Our data suggest that hemophilin is a hemophore andthat inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract