Involvement of HxuC outer membrane protein in utilization of hemoglobin byHaemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme fo r growth in vitro. A previous study from this laboratory indicated that non typeable Haemophilus influenzae (NTHI) strain N182 expressed three outer me mbrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one o f these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly be tter with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Co nstruction of an isogenic hgbA hgbB hgbC mutant revealed that the absence o f these three gene products did not affect the ability of NTHI N182 to util ize hemoglobin as a source of heme, although this mutant was severely impai red in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize he moglobin, indicating that the pathway for hemoglobin utilization in the abs ence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivatio n in this triple mutant of the hxuC gene, which encodes a predicted TonB-de pendent outer membrane protein previously shown to be involved in the utili zation of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acqui sition systems expressed by H. influenzae.