Heme sequestration by hemophilin from <i>Haemophilus haemolyticus</i> reduces respiratory tract colonization and infection with non-typeable <i>Haemophilus influenzae</i>

UNLABELLED: Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.