Abstract
Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.
Full Text
The Full Text of this article is available as a PDF (560.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Albritton W. L. Biology of Haemophilus ducreyi. Microbiol Rev. 1989 Dec;53(4):377–389. doi: 10.1128/mr.53.4.377-389.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- Cornelissen C. N., Biswas G. D., Tsai J., Paruchuri D. K., Thompson S. A., Sparling P. F. Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors. J Bacteriol. 1992 Sep;174(18):5788–5797. doi: 10.1128/jb.174.18.5788-5797.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cornelissen C. N., Sparling P. F. Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens. Mol Microbiol. 1994 Dec;14(5):843–850. doi: 10.1111/j.1365-2958.1994.tb01320.x. [DOI] [PubMed] [Google Scholar]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elkins C. Identification and purification of a conserved heme-regulated hemoglobin-binding outer membrane protein from Haemophilus ducreyi. Infect Immun. 1995 Apr;63(4):1241–1245. doi: 10.1128/iai.63.4.1241-1245.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gudmundsdottir A., Bell P. E., Lundrigan M. D., Bradbeer C., Kadner R. J. Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport. J Bacteriol. 1989 Dec;171(12):6526–6533. doi: 10.1128/jb.171.12.6526-6533.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hansen E. J., Latimer J. L., Thomas S. E., Helminen M., Albritton W. L., Radolf J. D. Use of electroporation to construct isogenic mutants of Haemophilus ducreyi. J Bacteriol. 1992 Aug;174(16):5442–5449. doi: 10.1128/jb.174.16.5442-5449.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henderson D. P., Payne S. M. Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins. J Bacteriol. 1994 Jun;176(11):3269–3277. doi: 10.1128/jb.176.11.3269-3277.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jarosik G. P., Sanders J. D., Cope L. D., Muller-Eberhard U., Hansen E. J. A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b. Infect Immun. 1994 Jun;62(6):2470–2477. doi: 10.1128/iai.62.6.2470-2477.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lee B. C. Iron sources for Haemophilus ducreyi. J Med Microbiol. 1991 Jun;34(6):317–322. doi: 10.1099/00222615-34-6-317. [DOI] [PubMed] [Google Scholar]
- Legrain M., Mazarin V., Irwin S. W., Bouchon B., Quentin-Millet M. J., Jacobs E., Schryvers A. B. Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2. Gene. 1993 Aug 16;130(1):73–80. doi: 10.1016/0378-1119(93)90348-7. [DOI] [PubMed] [Google Scholar]
- Lundrigan M. D., Kadner R. J. Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB. J Biol Chem. 1986 Aug 15;261(23):10797–10801. [PubMed] [Google Scholar]
- Morse S. A. Chancroid and Haemophilus ducreyi. Clin Microbiol Rev. 1989 Apr;2(2):137–157. doi: 10.1128/cmr.2.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nau C. D., Konisky J. Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene. J Bacteriol. 1989 Feb;171(2):1041–1047. doi: 10.1128/jb.171.2.1041-1047.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neilands J. B. Microbial iron compounds. Annu Rev Biochem. 1981;50:715–731. doi: 10.1146/annurev.bi.50.070181.003435. [DOI] [PubMed] [Google Scholar]
- Otto B. R., Verweij-van Vught A. M., MacLaren D. M. Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit Rev Microbiol. 1992;18(3):217–233. doi: 10.3109/10408419209114559. [DOI] [PubMed] [Google Scholar]
- Palmer K. L., Grass S., Munson R. S., Jr Identification of a hemolytic activity elaborated by Haemophilus ducreyi. Infect Immun. 1994 Jul;62(7):3041–3043. doi: 10.1128/iai.62.7.3041-3043.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pettersson A., van der Ley P., Poolman J. T., Tommassen J. Molecular characterization of the 98-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis. Infect Immun. 1993 Nov;61(11):4724–4733. doi: 10.1128/iai.61.11.4724-4733.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plummer F. A., Simonsen J. N., Cameron D. W., Ndinya-Achola J. O., Kreiss J. K., Gakinya M. N., Waiyaki P., Cheang M., Piot P., Ronald A. R. Cofactors in male-female sexual transmission of human immunodeficiency virus type 1. J Infect Dis. 1991 Feb;163(2):233–239. doi: 10.1093/infdis/163.2.233. [DOI] [PubMed] [Google Scholar]
- Seifert H. S., Chen E. Y., So M., Heffron F. Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Feb;83(3):735–739. doi: 10.1073/pnas.83.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stojiljkovic I., Hantke K. Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria. EMBO J. 1992 Dec;11(12):4359–4367. doi: 10.1002/j.1460-2075.1992.tb05535.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Struyvé M., Moons M., Tommassen J. Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J Mol Biol. 1991 Mar 5;218(1):141–148. doi: 10.1016/0022-2836(91)90880-f. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wasserheit J. N. Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other sexually transmitted diseases. Sex Transm Dis. 1992 Mar-Apr;19(2):61–77. [PubMed] [Google Scholar]
- Weinberg E. D. Iron and infection. Microbiol Rev. 1978 Mar;42(1):45–66. doi: 10.1128/mr.42.1.45-66.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Young K., Silver L. L. Leakage of periplasmic enzymes from envA1 strains of Escherichia coli. J Bacteriol. 1991 Jun;173(12):3609–3614. doi: 10.1128/jb.173.12.3609-3614.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]