Expression of the Haemophilus influenzae transferrin receptor is repressible by hemin but not elemental iron alone

The absolute requirement for elemental iron and the porphyrin nucleus for growth of Haemophilus influenzae led us to investigate the role of iron and hemin in regulation of expression of the H. influenzae transferrin receptor. H. influenzae type b strain HI689 was grown in brain heart infusion broth supplemented with β-NAD and either 10 or 0.1 μg of hemin ml^-1. Transferrin-binding ability was determined with a dot blot assay using human transferrin-horseradish peroxidase conjugate. Cells grown in media with 0.1 μg of hemin ml^-1 bound transferrin, but organisms grown in media with 10 μg ml^-1 did not. In hemin-restricted media, transferrin binding occurred despite addition of up to 10 mM ferric nitrate, ferric citrate, or ferric PP(i), whereas addition of 10 μg of hemoglobin ml^-1 repressed expression. The breadth of species distribution of this mode of regulation was determined with strains previously characterized by multilocus enzyme electrophoresis. When grown in hemin-restricted media, 24 of 28 type b strains and 52 of 57 serologically nontypeable strains exhibited transferrin binding, although none did so in hemin- and iron-sufficient media. Strain HI689 and serologically nontypeable strain HI1423 grown in heat-inactivated pooled normal human serum, human cerebrospinal fluid, or human breast milk exhibited transferrin binding. Growth in these fluids with 10 μg of added hemin ml^{- 1} abolished transferrin binding, whereas addition of 10 mM ferric nitrate did not. These data suggest that the transferrin receptor of H. influenzae is regulated by levels of hemin but not elemental iron alone and that this property is widely distributed among several major cloned families in the species.