AFFINITY, CONSERVATION, AND SURFACE EXPOSURE OF HEMOPEXIN-BINDING PROTEINS IN HAEMOPHILUS-INFLUENZAE

Haemophilus influenzae can acquire heme from hemopexin for use as a so urce of both essential porphyrin and iron. In classical ligand-binding studies, we observed time-dependent, saturable, and displaceable bind ing of human I-125-labelled hemopexin to intact cells of H. influenzae type b (Hib) strain 760705 grown in an iron-restricted medium. From t hese experiments, which demonstrate that hemopexin associates with a s ingle class of binding site, the affinities (K(d)s) and receptor numbe rs were calculated for heme-hemopexin (K-d, 205 nM; 3,200 receptors pe r cell) and apohemopexin (K-d, 392 nM; 4,400 receptors per cell). Thus , Hib expresses a specific hemopexin receptor which shows some prefere nce for the heme-protein complex. Affinity chromatography on hemopexin -Sepharose 4B of detergent-solubilized membranes from Hib strain 76070 5 results in the copurification of three proteins with molecular masse s of 57, 38, and 29 kDa. Trypsinization of whole cells of Hib 760705 a bolishes hemopexin binding and correlates with the disappearance of th e 57-kDa hemopexin-binding protein and appearance of a 52-kDa species which does not bind either hemopexin in ligand blot assays or a monocl onal antibody (MAbT11-30) raised against the 57-kDa protein. From immu noblotting assays and NH2-terminal amino acid sequence analysis, the 3 8-kDa protein isolated following hemopexin affinity chromatography was identified as the porin protein P2. These data, taken together with t he receptor-binding studies which support a single class of hemopexin- binding site, suggest that P2 and the 29-kDa protein function as acces sory proteins to the 57-kDa hemopexin-binding protein to facilitate th e uptake of heme from receptor-bound hemopexin. To determine whether h emopexin binding and the 57-kDa protein are conserved in Haemophilus s trains, whole-cell dot blots and immunoblots of the outer membrane pro teins prepared from strains belonging to each of 21 different Hib oute r membrane protein subtypes, six nontypeable strains, and five Haemoph ilus parainfluenzae strains were probed with either hemopexin or MAbT1 1-30. Only the H. parainfluenzae strains which lack the 57-kDa protein do not bind hemopexin. Since H. influenzae has also been shown to pro duce a soluble 100-kDa hemopexin-binding protein, cell-free culture su pernatants were also examined for the presence of this protein. Apart from Hib 760705 and H. parainfluenzae, the 100-kDa hemopexin-binding p rotein was detected in all the other Haemophilus strains. The abilitie s of Hib 760705 to both bind and acquire heme from hemopexin without e xpressing a 100-kDa soluble hemopexin-binding protein show that in str ain 760705, this 100-kDa protein is not essential for the utilization of heme from hemopexin.