Identification of a Genetic Locus of Haemophilus influenzae Type b Necessary for the Binding and Utilization of Heme Bound to Human Hemopexin

• Published in: Proceedings of the National Academy of Sciences - PNAS 1992-03, Vol.89 (5), p.1973-1977
• Main Authors: Hanson, Mark S., Pelzel, Sharon E., Latimer, Jo, Muller-Eberhard, Ursula, Hansen, Eric J.
• Format: Article
• Language: English
• Subjects: Antigens; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Biology; Carrier proteins; Cloning, Molecular; DNA; DNA, Bacterial - genetics; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Haemophilus; Haemophilus influenzae - genetics; Haemophilus influenzae - metabolism; Haemophilus influenzae - pathogenicity; Heme - metabolism; Hemoglobins; Hemopexin - metabolism; Humans; In Vitro Techniques; Medical research; Microbiology; Molecular Weight; Parasite hosts; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Pathogens; Plasmids; Recombinant Proteins - metabolism; Restriction Mapping
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.5.1973

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 ≈ 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.