Tristram, S. | Search bioRxiv

bioRxiv2020

In Vitro Probiotic Potential of Hemophilin-producing Strains of Haemophilus haemolyticus

Non-typeable Haemophilus influenzae (NTHi) is a leading causative organism of opportunistic respiratory tract infections, including otitis media and acute exacerbations of chronic obstructive pulmonary disease. Despite the enormous disease burden associated with NTHi infections, there are currently no effective prevention strategies, and the rapid development of antibiotic resistance is compromising treatment. We previously discovered Haemophilus haemolyticus (Hh) strains capable of producing haemophilin (HPL), a heme-binding protein that restricts NTHi growth by limiting its access to an essential growth factor, heme. Thus, these strains may have utility as a probiotic therapy against NTHi infection by limiting colonization, migration and subsequent infection in susceptible individuals. Here, we have assessed the feasibility of this approach by in vitro competition assays between NTHi and Hh strains with varying capacity to produce HPL. HPL-producing strains of Hh exhibited enhanced growth and consistently outcompeted NTHi compared to Hh strains unable to produce the protein. This competitive advantage was maintained over a period of six days, culminating in the complete eradication of NTHi. Expression analysis of HPL during competition coincided with the NTHi-inhibitory capacity of HPL-producers, confirming that inhibition was mediated by the presence of HPL. Together, results suggest that natural levels of HPL production by Hh are sufficient to limit NTHis access to heme, even under excess heme conditions unlikely to be encountered in vivo. Further investigation is required to determine the protective capacity of HPL-producers in vivo and their ability to interrupt NTHi colonization of host cells.

microbiology↗

bioRxiv2019

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

Many commensal bacteria and opportunistic pathogens scavenge heme from their environment. Pathogens and host are engaged in an arms race to control access to heme, but similar conflicts between bacterial species that might regulate pathogen colonisation are largely unknown. We show here that a commensal bacterium, Haemophilus haemolyticus, makes hemophilin, a heme-binding protein that not only allows the bacterium to effectively scavenge heme for its own growth, but also inhibits co-culture of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi), by heme starvation. Knockout of the hemophilin gene abrogates the ability of H. haemolyticus to inhibit NTHi and an x-ray crystal structure shows that hemophilin has a previously unreported heme-binding structure. The bound heme molecule is deeply buried and the heme iron atom is coordinated through a single histidine side chain. Biochemical characterization shows that this arrangement allows heme to be captured in the ferrous or ferric state, and with small ferrous or ferric heme-ligands bound, suggesting hemophilin could function over in a wide range of physiological conditions. Our data raise the possibility that competition for heme between commensal and pathogenic bacteria can influence bacterial colonisation, and therefore disease likelihood, and suggest that strains of H. haemolyticus that overproduce hemophilin might have therapeutic uses in reducing colonisation and subsequent opportunistic infection by NTHi.

biochemistry↗