Abstract
The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.
Full Text
The Full Text of this article is available as a PDF (508.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Badger J. L., Miller V. L. Role of RpoS in survival of Yersinia enterocolitica to a variety of environmental stresses. J Bacteriol. 1995 Sep;177(18):5370–5373. doi: 10.1128/jb.177.18.5370-5373.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bottone E. J. Yersinia enterocolitica: a panoramic view of a charismatic microorganism. CRC Crit Rev Microbiol. 1977;5(2):211–241. doi: 10.3109/10408417709102312. [DOI] [PubMed] [Google Scholar]
- Bowe F., O'Gaora P., Maskell D., Cafferkey M., Dougan G. Virulence, persistence, and immunogenicity of Yersinia enterocolitica O:8 aroA mutants. Infect Immun. 1989 Oct;57(10):3234–3236. doi: 10.1128/iai.57.10.3234-3236.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brenner S. The molecular evolution of genes and proteins: a tale of two serines. Nature. 1988 Aug 11;334(6182):528–530. doi: 10.1038/334528a0. [DOI] [PubMed] [Google Scholar]
- Bäumler A. J., Kusters J. G., Stojiljkovic I., Heffron F. Salmonella typhimurium loci involved in survival within macrophages. Infect Immun. 1994 May;62(5):1623–1630. doi: 10.1128/iai.62.5.1623-1630.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carter P. B., Collins F. M. Experimental Yersinia enterocolitica infection in mice: kinetics of growth. Infect Immun. 1974 May;9(5):851–857. doi: 10.1128/iai.9.5.851-857.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carter P. B. Pathogenecity of Yersinia enterocolitica for mice. Infect Immun. 1975 Jan;11(1):164–170. doi: 10.1128/iai.11.1.164-170.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chatfield S. N., Strahan K., Pickard D., Charles I. G., Hormaeche C. E., Dougan G. Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model. Microb Pathog. 1992 Feb;12(2):145–151. doi: 10.1016/0882-4010(92)90117-7. [DOI] [PubMed] [Google Scholar]
- China B., N'Guyen B. T., de Bruyere M., Cornelis G. R. Role of YadA in resistance of Yersinia enterocolitica to phagocytosis by human polymorphonuclear leukocytes. Infect Immun. 1994 Apr;62(4):1275–1281. doi: 10.1128/iai.62.4.1275-1281.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cornelis G. R., Biot T., Lambert de Rouvroit C., Michiels T., Mulder B., Sluiters C., Sory M. P., Van Bouchaute M., Vanooteghem J. C. The Yersinia yop regulon. Mol Microbiol. 1989 Oct;3(10):1455–1459. doi: 10.1111/j.1365-2958.1989.tb00129.x. [DOI] [PubMed] [Google Scholar]
- Cornelis G., Laroche Y., Balligand G., Sory M. P., Wauters G. Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev Infect Dis. 1987 Jan-Feb;9(1):64–87. doi: 10.1093/clinids/9.1.64. [DOI] [PubMed] [Google Scholar]
- Cover T. L., Aber R. C. Yersinia enterocolitica. N Engl J Med. 1989 Jul 6;321(1):16–24. doi: 10.1056/NEJM198907063210104. [DOI] [PubMed] [Google Scholar]
- Davies K. J., Lin S. W. Degradation of oxidatively denatured proteins in Escherichia coli. Free Radic Biol Med. 1988;5(4):215–223. doi: 10.1016/0891-5849(88)90015-9. [DOI] [PubMed] [Google Scholar]
- Elzer P. H., Phillips R. W., Kovach M. E., Peterson K. M., Roop R. M., 2nd Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant. Infect Immun. 1994 Oct;62(10):4135–4139. doi: 10.1128/iai.62.10.4135-4139.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Everest P., Frankel G., Li J., Lund P., Chatfield S., Dougan G. Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian cells. FEMS Microbiol Lett. 1995 Feb 1;126(1):97–101. doi: 10.1111/j.1574-6968.1995.tb07398.x. [DOI] [PubMed] [Google Scholar]
- Forsberg A., Rosqvist R., Wolf-Watz H. Regulation and polarized transfer of the Yersinia outer proteins (Yops) involved in antiphagocytosis. Trends Microbiol. 1994 Jan;2(1):14–19. doi: 10.1016/0966-842x(94)90339-5. [DOI] [PubMed] [Google Scholar]
- Forsberg A., Wolf-Watz H. Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression. J Bacteriol. 1990 Mar;172(3):1547–1555. doi: 10.1128/jb.172.3.1547-1555.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Galyov E. E., Håkansson S., Forsberg A., Wolf-Watz H. A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature. 1993 Feb 25;361(6414):730–732. doi: 10.1038/361730a0. [DOI] [PubMed] [Google Scholar]
- Grinter N. J. A broad-host-range cloning vector transposable to various replicons. Gene. 1983 Jan-Feb;21(1-2):133–143. doi: 10.1016/0378-1119(83)90155-5. [DOI] [PubMed] [Google Scholar]
- Hartland E. L., Green S. P., Phillips W. A., Robins-Browne R. M. Essential role of YopD in inhibition of the respiratory burst of macrophages by Yersinia enterocolitica. Infect Immun. 1994 Oct;62(10):4445–4453. doi: 10.1128/iai.62.10.4445-4453.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heesemann J., Gaede K., Autenrieth I. B. Experimental Yersinia enterocolitica infection in rodents: a model for human yersiniosis. APMIS. 1993 Jun;101(6):417–429. [PubMed] [Google Scholar]
- Holmström A., Rosqvist R., Wolf-Watz H., Forsberg A. Virulence plasmid-encoded YopK is essential for Yersinia pseudotuberculosis to cause systemic infection in mice. Infect Immun. 1995 Jun;63(6):2269–2276. doi: 10.1128/iai.63.6.2269-2276.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson K. S., Charles I. G., Dougan G., Miller I. A., Pickard D., O'Goara P., Costa G., Ali T., Hormaeche C. E. The role of a stress-response protein in bacterial virulence. Res Microbiol. 1990 Sep-Oct;141(7-8):823–825. doi: 10.1016/0923-2508(90)90116-8. [DOI] [PubMed] [Google Scholar]
- Johnson K., Charles I., Dougan G., Pickard D., O'Gaora P., Costa G., Ali T., Miller I., Hormaeche C. The role of a stress-response protein in Salmonella typhimurium virulence. Mol Microbiol. 1991 Feb;5(2):401–407. doi: 10.1111/j.1365-2958.1991.tb02122.x. [DOI] [PubMed] [Google Scholar]
- Lipinska B., Fayet O., Baird L., Georgopoulos C. Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. J Bacteriol. 1989 Mar;171(3):1574–1584. doi: 10.1128/jb.171.3.1574-1584.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lipinska B., Sharma S., Georgopoulos C. Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32-independent mechanism of heat-inducible transcription. Nucleic Acids Res. 1988 Nov 11;16(21):10053–10067. doi: 10.1093/nar/16.21.10053. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lipinska B., Zylicz M., Georgopoulos C. The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase. J Bacteriol. 1990 Apr;172(4):1791–1797. doi: 10.1128/jb.172.4.1791-1797.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mukkur T. K., McDowell G. H., Stocker B. A., Lascelles A. K. Protection against experimental salmonellosis in mice and sheep by immunisation with aromatic-dependent Salmonella typhimurium. J Med Microbiol. 1987 Aug;24(1):11–19. doi: 10.1099/00222615-24-1-11. [DOI] [PubMed] [Google Scholar]
- O'Callaghan D., Maskell D., Liew F. Y., Easmon C. S., Dougan G. Characterization of aromatic- and purine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB/c mice. Infect Immun. 1988 Feb;56(2):419–423. doi: 10.1128/iai.56.2.419-423.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pepe J. C., Badger J. L., Miller V. L. Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene. Mol Microbiol. 1994 Jan;11(1):123–135. doi: 10.1111/j.1365-2958.1994.tb00295.x. [DOI] [PubMed] [Google Scholar]
- Pepe J. C., Wachtel M. R., Wagar E., Miller V. L. Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection. Infect Immun. 1995 Dec;63(12):4837–4848. doi: 10.1128/iai.63.12.4837-4848.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riley G., Toma S. Detection of pathogenic Yersinia enterocolitica by using congo red-magnesium oxalate agar medium. J Clin Microbiol. 1989 Jan;27(1):213–214. doi: 10.1128/jcm.27.1.213-214.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roop R. M., 2nd, Fletcher T. W., Sriranganathan N. M., Boyle S. M., Schurig G. G. Identification of an immunoreactive Brucella abortus HtrA stress response protein homolog. Infect Immun. 1994 Mar;62(3):1000–1007. doi: 10.1128/iai.62.3.1000-1007.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosqvist R., Forsberg A., Rimpiläinen M., Bergman T., Wolf-Watz H. The cytotoxic protein YopE of Yersinia obstructs the primary host defence. Mol Microbiol. 1990 Apr;4(4):657–667. doi: 10.1111/j.1365-2958.1990.tb00635.x. [DOI] [PubMed] [Google Scholar]
- Ruiz-Bravo A., Jimenez-Valera M., Sampedro A., Moreno E. Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages. FEMS Immunol Med Microbiol. 1994 Oct;9(4):317–324. doi: 10.1111/j.1574-695X.1994.tb00367.x. [DOI] [PubMed] [Google Scholar]
- Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
- Seol J. H., Woo S. K., Jung E. M., Yoo S. J., Lee C. S., Kim K. J., Tanaka K., Ichihara A., Ha D. B., Chung C. H. Protease Do is essential for survival of Escherichia coli at high temperatures: its identity with the htrA gene product. Biochem Biophys Res Commun. 1991 Apr 30;176(2):730–736. doi: 10.1016/s0006-291x(05)80245-1. [DOI] [PubMed] [Google Scholar]
- Strahan K., Chatfield S. N., Tite J., Dougan G., Hormaeche C. E. Impaired resistance to infection does not increase the virulence of Salmonella htrA live vaccines for mice. Microb Pathog. 1992 Apr;12(4):311–317. doi: 10.1016/0882-4010(92)90049-t. [DOI] [PubMed] [Google Scholar]
- Straley S. C., Perry R. D. Environmental modulation of gene expression and pathogenesis in Yersinia. Trends Microbiol. 1995 Aug;3(8):310–317. doi: 10.1016/s0966-842x(00)88960-x. [DOI] [PubMed] [Google Scholar]
- Strauch K. L., Beckwith J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1576–1580. doi: 10.1073/pnas.85.5.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strauch K. L., Johnson K., Beckwith J. Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J Bacteriol. 1989 May;171(5):2689–2696. doi: 10.1128/jb.171.5.2689-2696.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tatum F. M., Cheville N. F., Morfitt D. Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice. Microb Pathog. 1994 Jul;17(1):23–36. doi: 10.1006/mpat.1994.1049. [DOI] [PubMed] [Google Scholar]
- Une T. Studies on the pathogenicity of Yersinia enterocolitica. I. Experimental infection in rabbits. Microbiol Immunol. 1977;21(7):341–363. [PubMed] [Google Scholar]
- Une T. Studies on the pathogenicity of Yersinia enterocolitica. II. Interaction with cultured cells in vitro. Microbiol Immunol. 1977;21(7):365–377. doi: 10.1111/j.1348-0421.1977.tb00301.x. [DOI] [PubMed] [Google Scholar]
- Wierenga R. K., Hol W. G. Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature. 1983 Apr 28;302(5911):842–844. doi: 10.1038/302842a0. [DOI] [PubMed] [Google Scholar]
- Wren B. W., Colby S. M., Cubberley R. R., Pallen M. J. Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):287–291. doi: 10.1016/0378-1097(92)90042-m. [DOI] [PubMed] [Google Scholar]
- Wren B. W., Henderson J., Ketley J. M. A PCR-based strategy for the rapid construction of defined bacterial deletion mutants. Biotechniques. 1994 Jun;16(6):994–996. [PubMed] [Google Scholar]
- Wren B. W., Tabaqchali S. Detection of pathogenic Yersinia enterocolitica by the polymerase chain reaction. Lancet. 1990 Sep 15;336(8716):693–693. doi: 10.1016/0140-6736(90)92191-j. [DOI] [PubMed] [Google Scholar]
- Zhang L., Skurnik M. Isolation of an R- M+ mutant of Yersinia enterocolitica serotype O:8 and its application in construction of rough mutants utilizing mini-Tn5 derivatives and lipopolysaccharide-specific phage. J Bacteriol. 1994 Mar;176(6):1756–1760. doi: 10.1128/jb.176.6.1756-1760.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]