Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two he moglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this th ird hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC . Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC trip le mutant constructed by insertional mutagenesis showed a reduced ability t o use the hemoglobin-haptoglobin complex but was unaltered in the ability t o use hemoglobin. A second class of mutants was constructed in which the en tire structural gene of each of the three proteins was deleted. The hgpA hg pB hgpC complete-deletion triple mutant was unable to utilize the hemoglobi n-haptoglobin complex and showed a reduced ability to use hemoglobin. We ha ve identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in H aemophilus influenzae. Any one of the three proteins is sufficient to suppo rt growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilizat ion. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.