Haem at the interface between pathogenic and commensal bacterial species in the human respiratory tract

Non-typeable Haemophilus influenzae (NTHi) is an important opportunistic pathogen of the human respiratory tract that has proved recalcitrant to vaccine development and shows increasing prevalence of antibiotic resistance, prompting us to search for alternative anti-microbial strategies. Haemophilus haemolyticus is closely related to NTHi, and also colonises the respiratory tract, but is recognised as a non-pathogenic commensal species. We identified two H. haemolyticusisolates secreting a 27-kDa protein that inhibited the growth of NTHi in vitro, but did not inhibit a range of other respiratory flora that were tested, suggesting a level of species-specificity against NTHi. A gene knockout established that the gene product of interest was responsible for inhibitory activity. Spectroscopic and x-ray crystallographic analysis of the recombinant protein identified a haem binding site. The protein shares structural features with some non-haem iron scavenging proteins, but the haem-binding site is unique. Insights into the biological function, including NTHi inhibitory actions, of this protein, based on structure, biochemistry and bioactivity assays are presented. The work is ongoing in the context that strains of H. haemolyticus might be developed as respiratory probiotics to combat colonisation and infection with NTHi.