THE 100 KDA HEME-HEMOPEXIN-BINDING PROTEIN OF HAEMOPHILUS-INFLUENZAE - STRUCTURE AND LOCALIZATION

All Haemophilus influenzae strains have an absolute requirement for ex ogenously supplied haem for aerobic growth. A majority of strains of H . influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, de signated HxuA, was originally detected on the Hib cell surface. Monocl onal antibody (mAb)-based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin-binding activity in a direct b inding assay. Nucleotide sequence analysis of the hxuA gene from Hib s train DL42, together with N-terminal amino acid analysis of HxuA prote in purified from Hib culture supernatant, revealed that this protein w as synthesized as a 101 kDa precursor with a leader peptide that was r emoved to yield a 99 kDa protein. Southern blot analysis of chromosoma l DNA from four Hib and four non-typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains show ed that all four strains released into culture supernatant a haem:haem opexin-binding protein that migrated in SDS-PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence ana lysis of this NTHI gene revealed that it encoded a protein with 87% id entity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system i s conserved among H. influenzae strains.