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

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.