Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b

Abstract

Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two hemopexin receptors, the expression of which is determined by the prevailing growth environment.

Haem release from haemopexin by HxuA allows Haemophilus influenzae to escape host nutritional immunity

“…Furthermore, the HxuA-haemopexin complex lacks the high affinity for haem of haemopexin alone, and our experiments set an upper limit for the potential affinity of the HxuAhaemopexin complex for haem (below 10 6 M -1 , much lower than that of human serum albumin). HxuA interacts very strongly with haemopexin, as shown in previous studies (Wong et al, 1994;, and we have shown that HxuA forms high-affinity stoichiometric complexes with both apo-and haem-haemopexin. As the haem environment is modified by the formation of a complex between HxuA and haem-haemopexin, the affinity constant determined by ITC for this interaction is only apparent, as it takes into account both the protein-protein interaction and the change in haem environment.…”

Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae

“…Recent studies have identi¢ed an additional heme: hemopexin binding complex that is regulated by ambient levels of iron [167]. In this complex, the receptor is a 57-kDa protein that is anchored in the outer membrane.…”

Molecular determinants of the pathogenesis of disease due to non-typableHaemophilus influenzae

“…Recent studies have identified an additional heme:hemopexin binding complex that is regulated by ambient levels of iron [167]. In this complex, the receptor is a 57‐kDa protein that is anchored in the outer membrane.…”