Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemopexin

Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1973-7. doi: 10.1073/pnas.89.5.1973.

Abstract

The mechanism(s) used by Haemophilus influenzae to acquire the essential nutrient heme from its human host has not been elucidated. The heme carried by the high-affinity serum protein hemopexin is one potential source of this micronutrient in vivo. A colony-blot assay revealed that heme-human hemopexin-binding activity was shared among most capsular serotype b strains of H. influenzae but was uncommon among other strains. We have identified a recombinant clone binding heme-human hemopexin from a H. influenzae type b (Hib) genomic library expressed in Escherichia coli. Both the Hib strain and the heme-hemopexin-binding clone expressed a polypeptide of approximately 100 kDa that bound radiolabeled heme-hemopexin. Oligonucleotide linker insertion mutagenesis of the plasmid DNA from this recombinant clone was used to confirm that expression of the 100-kDa protein correlated with the heme-hemopexin-binding activity. Exchange of one of these mutant alleles into the Hib chromosome eliminated expression of both the 100-kDa protein and the heme-hemopexin-binding activity. Furthermore, this Hib mutant was unable to utilize heme-human hemopexin as a heme source.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Cloning, Molecular
  • DNA, Bacterial / genetics
  • Genes, Bacterial*
  • Haemophilus influenzae / genetics*
  • Haemophilus influenzae / metabolism
  • Haemophilus influenzae / pathogenicity
  • Heme / metabolism*
  • Hemopexin / metabolism*
  • Humans
  • In Vitro Techniques
  • Molecular Weight
  • Recombinant Proteins / metabolism
  • Restriction Mapping

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Recombinant Proteins
  • Heme
  • Hemopexin