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

Abstract Haemophilus haemolyticus is a commensal bacterium of humans and occupies the same respiratory tract niche as the pathogenic bacterium, non-typeable Haemophilus influenzae (NTHi). Previously we showed that an isolate of H. haemolyticus secretes a protein that inhibits the growth of NTHi in culture. Here, we purify the H. haemolyticus NTHi-inhibitory protein, identify the gene using mass spectrometry and proteomics, and show that the recombinant protein produced in E. coli carrys a bound heme molecule, leading us to name it hemophilin. Recombinant hemophilin has NTHi-inhibitory activity and a gene knockout in H. haemolyticus confirms that hemophilin is the source of NTHi-inhibitory activity in the native organism. An x-ray crystal structure shows that hemophilin has a topology similar to bacterial proteins that bind human transferrin, hemoglobin, complement factor H and heparin, but none of these proteins bind heme, and no heme-binding domain with similarity to hemophilin could be detected by structure-or sequence-based searches. The heme occupies a pocket that is protected from solvent, with the heme iron atom coordinated by the side chain of His119. Hemophilin knockout bacteria show a limited capacity to utilise ferric heme to support growth, compared to the parent strain. The data suggest that hemophilin is a hemophore and that inhibition of NTHi growth occurs by heme starvation, raising the possibility that competition for heme between some commensal and pathogenic bacteria might influence bacterial colonisation and therefore disease outcome. Significance statementNon-typeable Haemophilus influenzae (NTHi) are human pathogens that reside in the upper respiratory tract and require heme for growth. There is currently no effective vaccination against this pathogen, and antibiotic control is becoming less effective. Haemophilus haemolyticus is a commensal of the upper respiratory tract, and is also a heme auxotroph. Our study suggests that production of hemophilin might allow some strains of H. haemolyticus to compete more effectively with NTHi. Lower colonisation density may result in less frequent opportunistic NTHi infections and provide an alternative means for preventing infection.