Abstract
We previously reported the cloning and sequencing of genes designated hmw1 and hmw2 from a prototype nontypeable Haemophilus influenzae strain. The genes encode proteins which are related to filamentous hemagglutinin of Bordetella pertussis and promote attachment of the nontypeable H. influenzae strain to human epithelial cells (J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). Subcloning studies suggested that correct processing of these high-molecular-weight proteins required the products of additional downstream genes. In the present study we analyzed the 3'-flanking regions of the hmw1A and hmw2A structural genes and found that both genes are flanked by two additional downstream open reading frames (ORFs), designated B and C, respectively. The B ORFs are 1,635 bp long. Their derived amino acid sequences are 99% identical and demonstrate similarity to the derived amino acid sequences of two genes that encode proteins required for secretion and activation of hemolysins of Proteus mirabilis and Serratia marcescens. The C ORFs are 1,950 bp long, and their derived amino acid sequences are 96% identical. In Escherichia coli transformants, interruption of the hmw1C or both the hmw1B and hmw1C genes resulted in defective processing of the hmw1A structural gene product and loss of the ability of the transformants to adhere to human epithelial cells. The precise interactions of the proteins encoded by these gene clusters are yet to be defined, but their elucidation may further our understanding of the biology of nontypeable H. influenzae bacteria and the interaction of these organisms with the human host.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barenkamp S. J., Bodor F. F. Development of serum bactericidal activity following nontypable Haemophilus influenzae acute otitis media. Pediatr Infect Dis J. 1990 May;9(5):333–339. doi: 10.1097/00006454-199005000-00006. [DOI] [PubMed] [Google Scholar]
- Barenkamp S. J., Leininger E. Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis. Infect Immun. 1992 Apr;60(4):1302–1313. doi: 10.1128/iai.60.4.1302-1313.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barnes W. M. Sequencing DNA with dideoxyribonucleotides as chain terminators: hints and strategies for big projects. Methods Enzymol. 1987;152:538–556. doi: 10.1016/0076-6879(87)52060-2. [DOI] [PubMed] [Google Scholar]
- Braun V., Hobbie S., Ondraczek R. Serratia marcescens forms a new type of cytolysin. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):299–305. doi: 10.1111/j.1574-6968.1992.tb14056.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]
- Domenighini M., Relman D., Capiau C., Falkow S., Prugnola A., Scarlato V., Rappuoli R. Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor. Mol Microbiol. 1990 May;4(5):787–800. doi: 10.1111/j.1365-2958.1990.tb00649.x. [DOI] [PubMed] [Google Scholar]
- Ely S., Tippett J., Kroll J. S., Moxon E. R. Mutations affecting expression and maintenance of genes encoding the serotype b capsule of Haemophilus influenzae. J Bacteriol. 1986 Jul;167(1):44–48. doi: 10.1128/jb.167.1.44-48.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
- Moffatt B. A., Studier F. W. T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell. 1987 Apr 24;49(2):221–227. doi: 10.1016/0092-8674(87)90563-0. [DOI] [PubMed] [Google Scholar]
- Noel G. J., Barenkamp S. J., St Geme J. W., 3rd, Haining W. N., Mosser D. M. High-molecular-weight surface-exposed proteins of Haemophilus influenzae mediate binding to macrophages. J Infect Dis. 1994 Feb;169(2):425–429. doi: 10.1093/infdis/169.2.425. [DOI] [PubMed] [Google Scholar]
- Poole K., Schiebel E., Braun V. Molecular characterization of the hemolysin determinant of Serratia marcescens. J Bacteriol. 1988 Jul;170(7):3177–3188. doi: 10.1128/jb.170.7.3177-3188.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Relman D. A., Domenighini M., Tuomanen E., Rappuoli R., Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2637–2641. doi: 10.1073/pnas.86.8.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schiebel E., Schwarz H., Braun V. Subcellular location and unique secretion of the hemolysin of Serratia marcescens. J Biol Chem. 1989 Sep 25;264(27):16311–16320. [PubMed] [Google Scholar]
- Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
- St Geme J. W., 3rd, Falkow S., Barenkamp S. J. High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2875–2879. doi: 10.1073/pnas.90.7.2875. [DOI] [PMC free article] [PubMed] [Google Scholar]
- St Geme J. W., 3rd, Falkow S. Haemophilus influenzae adheres to and enters cultured human epithelial cells. Infect Immun. 1990 Dec;58(12):4036–4044. doi: 10.1128/iai.58.12.4036-4044.1990. [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]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uphoff T. S., Welch R. A. Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB). J Bacteriol. 1990 Mar;172(3):1206–1216. doi: 10.1128/jb.172.3.1206-1216.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wallace R. J., Jr, Musher D. M., Septimus E. J., McGowan J. E., Jr, Quinones F. J., Wiss K., Vance P. H., Trier P. A. Haemophilus influenzae infections in adults: characterization of strains by serotypes, biotypes, and beta-lactamase production. J Infect Dis. 1981 Aug;144(2):101–106. doi: 10.1093/infdis/144.2.101. [DOI] [PubMed] [Google Scholar]
- Willems R. J., Geuijen C., van der Heide H. G., Renauld G., Bertin P., van den Akker W. M., Locht C., Mooi F. R. Mutational analysis of the Bordetella pertussis fim/fha gene cluster: identification of a gene with sequence similarities to haemolysin accessory genes involved in export of FHA. Mol Microbiol. 1994 Jan;11(2):337–347. doi: 10.1111/j.1365-2958.1994.tb00314.x. [DOI] [PubMed] [Google Scholar]