Abstract
Brucella abortus is a facultative, intracellular, pathogenic bacterium that replicates within macrophages and resists macrophage microbicidal mechanisms. To study gene expression and to elucidate the defense mechanisms used by B. abortus to resist destruction within macrophages, protein synthesis by B. abortus was examined by pulse-labeling techniques during intracellular growth within J774A.1, a macrophage-like cell line. Prominent changes observed include increased synthesis of Brucella proteins with estimated molecular masses of 62, 28, 24, and 17 kDa. The 62-kDa protein was identified by immunoprecipitation analysis as Hsp62, a GroEL homolog. A protein of 60 kDa was expressed during acid shock and may represent a modified form of Hsp62. The 28- and 17-kDa proteins have not been observed under any in vitro stress condition and presumably represent macrophage-specific induction. The 24-kDa protein comigrates with an unidentified protein induced by acid shock, designated Asp24. Expression of Asp24 is optimal at pH values below 4.0 and within the first 3 h following a shift from pH 7.3 to 3.8. This corresponds directly with a period of optimal bacterial survival at a reduced pH and suggests an active role for this protein in resistance to such environments. The identification of these gene products and the mechanisms controlling their expression is an important step in understanding the resistance of Brucella spp. to intracellular destruction within macrophages.
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- Abu Kwaik Y., Eisenstein B. I., Engleberg N. C. Phenotypic modulation by Legionella pneumophila upon infection of macrophages. Infect Immun. 1993 Apr;61(4):1320–1329. doi: 10.1128/iai.61.4.1320-1329.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Alpuche Aranda C. M., Swanson J. A., Loomis W. P., Miller S. I. Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10079–10083. doi: 10.1073/pnas.89.21.10079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Auger E. A., Redding K. E., Plumb T., Childs L. C., Meng S. Y., Bennett G. N. Construction of lac fusions to the inducible arginine- and lysine decarboxylase genes of Escherichia coli K12. Mol Microbiol. 1989 May;3(5):609–620. doi: 10.1111/j.1365-2958.1989.tb00208.x. [DOI] [PubMed] [Google Scholar]
- Booth I. R. Regulation of cytoplasmic pH in bacteria. Microbiol Rev. 1985 Dec;49(4):359–378. doi: 10.1128/mr.49.4.359-378.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bounous D. I., Enright F. M., Gossett K. A., Berry C. M. Phagocytosis, killing, and oxidant production by bovine monocyte-derived macrophages upon exposure to Brucella abortus strain 2308. Vet Immunol Immunopathol. 1993 Aug;37(3-4):243–256. doi: 10.1016/0165-2427(93)90197-c. [DOI] [PubMed] [Google Scholar]
- Buchmeier N. A., Heffron F. Induction of Salmonella stress proteins upon infection of macrophages. Science. 1990 May 11;248(4956):730–732. doi: 10.1126/science.1970672. [DOI] [PubMed] [Google Scholar]
- Buchmeier N. A., Lipps C. J., So M. Y., Heffron F. Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages. Mol Microbiol. 1993 Mar;7(6):933–936. doi: 10.1111/j.1365-2958.1993.tb01184.x. [DOI] [PubMed] [Google Scholar]
- Campbell G. A., Adams L. G., Sowa B. A. Mechanisms of binding of Brucella abortus to mononuclear phagocytes from cows naturally resistant or susceptible to brucellosis. Vet Immunol Immunopathol. 1994 Jun;41(3-4):295–306. doi: 10.1016/0165-2427(94)90103-1. [DOI] [PubMed] [Google Scholar]
- Campbell G. A., Adams L. G. The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus. Vet Immunol Immunopathol. 1992 Nov;34(3-4):291–305. doi: 10.1016/0165-2427(92)90171-l. [DOI] [PubMed] [Google Scholar]
- Carazo J. M., Marco S., Abella G., Carrascosa J. L., Secilla J. P., Muyal M. Electron microscopy study of GroEL chaperonin: different views of the aggregate appear as a function of cell growth temperature. J Struct Biol. 1991 Jun;106(3):211–220. doi: 10.1016/1047-8477(91)90071-4. [DOI] [PubMed] [Google Scholar]
- Detilleux P. G., Deyoe B. L., Cheville N. F. Effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells. Am J Vet Res. 1991 Oct;52(10):1658–1664. [PubMed] [Google Scholar]
- Evans D. J., Jr, Evans D. G., Engstrand L., Graham D. Y. Urease-associated heat shock protein of Helicobacter pylori. Infect Immun. 1992 May;60(5):2125–2127. doi: 10.1128/iai.60.5.2125-2127.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fang F. C., Libby S. J., Buchmeier N. A., Loewen P. C., Switala J., Harwood J., Guiney D. G. The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11978–11982. doi: 10.1073/pnas.89.24.11978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foster J. W. Salmonella acid shock proteins are required for the adaptive acid tolerance response. J Bacteriol. 1991 Nov;173(21):6896–6902. doi: 10.1128/jb.173.21.6896-6902.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foster J. W. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins. J Bacteriol. 1993 Apr;175(7):1981–1987. doi: 10.1128/jb.175.7.1981-1987.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Govezensky D., Greener T., Segal G., Zamir A. Involvement of GroEL in nif gene regulation and nitrogenase assembly. J Bacteriol. 1991 Oct;173(20):6339–6346. doi: 10.1128/jb.173.20.6339-6346.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guglielmi G., Mazodier P., Thompson C. J., Davies J. A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus. J Bacteriol. 1991 Nov;173(22):7374–7381. doi: 10.1128/jb.173.22.7374-7381.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harmon B. G., Adams L. G., Frey M. Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages. Am J Vet Res. 1988 Jul;49(7):1092–1097. [PubMed] [Google Scholar]
- Heyde M., Portalier R. Acid shock proteins of Escherichia coli. FEMS Microbiol Lett. 1990 May;57(1-2):19–26. doi: 10.1016/0378-1097(90)90406-g. [DOI] [PubMed] [Google Scholar]
- Heyde M., Portalier R. Regulation of major outer membrane porin proteins of Escherichia coli K 12 by pH. Mol Gen Genet. 1987 Jul;208(3):511–517. doi: 10.1007/BF00328148. [DOI] [PubMed] [Google Scholar]
- Hickey E. W., Hirshfield I. N. Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium. Appl Environ Microbiol. 1990 Apr;56(4):1038–1045. doi: 10.1128/aem.56.4.1038-1045.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiang X., Baldwin C. L. Effects of cytokines on intracellular growth of Brucella abortus. Infect Immun. 1993 Jan;61(1):124–134. doi: 10.1128/iai.61.1.124-134.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kong T. H., Coates A. R., Butcher P. D., Hickman C. J., Shinnick T. M. Mycobacterium tuberculosis expresses two chaperonin-60 homologs. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2608–2612. doi: 10.1073/pnas.90.7.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kugel H., Mayer A., Kirst G. O., Leibfritz D. The energy requirements of pH homoeostasis define the limits of pH regulation--a model. Biochim Biophys Acta. 1990 Aug 13;1054(1):33–40. doi: 10.1016/0167-4889(90)90202-o. [DOI] [PubMed] [Google Scholar]
- Lehel C., Los D., Wada H., Györgyei J., Horváth I., Kovács E., Murata N., Vigh L. A second groEL-like gene, organized in a groESL operon is present in the genome of Synechocystis sp. PCC 6803. J Biol Chem. 1993 Jan 25;268(3):1799–1804. [PubMed] [Google Scholar]
- Leyer G. J., Johnson E. A. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl Environ Microbiol. 1993 Jun;59(6):1842–1847. doi: 10.1128/aem.59.6.1842-1847.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin J., Adams L. G., Ficht T. A. Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins. Infect Immun. 1992 Jun;60(6):2425–2431. doi: 10.1128/iai.60.6.2425-2431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marco S., Valpuesta J. M., Rivas G., Andrés G., San Martín C., Carrascosa J. L. A structural model for the GroEL chaperonin. FEMS Microbiol Lett. 1993 Feb 1;106(3):301–308. doi: 10.1111/j.1574-6968.1993.tb05980.x. [DOI] [PubMed] [Google Scholar]
- Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
- Miller S. I. PhoP/PhoQ: macrophage-specific modulators of Salmonella virulence? Mol Microbiol. 1991 Sep;5(9):2073–2078. doi: 10.1111/j.1365-2958.1991.tb02135.x. [DOI] [PubMed] [Google Scholar]
- Nauman R. K., Silverman D. J., Voelz H. Ribosomal helices: formation in Escherichia coli during acidic growth. J Bacteriol. 1971 Jul;107(1):358–360. doi: 10.1128/jb.107.1.358-360.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nicoletti P. The epidemiology of bovine brucellosis. Adv Vet Sci Comp Med. 1980;24:69–98. [PubMed] [Google Scholar]
- O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
- PAYNE J. M. The pathogenesis of experimental brucellosis in the pregnant cow. J Pathol Bacteriol. 1959 Oct;78:447–463. doi: 10.1002/path.1700780211. [DOI] [PubMed] [Google Scholar]
- Raja N., Goodson M., Chui W. C., Smith D. G., Rowbury R. J. Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer. J Appl Bacteriol. 1991 Jan;70(1):59–65. doi: 10.1111/j.1365-2672.1991.tb03787.x. [DOI] [PubMed] [Google Scholar]
- Riley L. K., Robertson D. C. Ingestion and intracellular survival of Brucella abortus in human and bovine polymorphonuclear leukocytes. Infect Immun. 1984 Oct;46(1):224–230. doi: 10.1128/iai.46.1.224-230.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rinke de Wit T. F., Bekelie S., Osland A., Miko T. L., Hermans P. W., van Soolingen D., Drijfhout J. W., Schöningh R., Janson A. A., Thole J. E. Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES. Mol Microbiol. 1992 Jul;6(14):1995–2007. doi: 10.1111/j.1365-2958.1992.tb01372.x. [DOI] [PubMed] [Google Scholar]
- Rowbury R. J., Goodson M., Wallace A. D. The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli. J Appl Bacteriol. 1992 Mar;72(3):233–243. doi: 10.1111/j.1365-2672.1992.tb01829.x. [DOI] [PubMed] [Google Scholar]
- Schellhorn H. E., Stones V. L. Regulation of katF and katE in Escherichia coli K-12 by weak acids. J Bacteriol. 1992 Jul;174(14):4769–4776. doi: 10.1128/jb.174.14.4769-4776.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sherman MYu, Goldberg A. L. Heat shock in Escherichia coli alters the protein-binding properties of the chaperonin groEL by inducing its phosphorylation. Nature. 1992 May 14;357(6374):167–169. doi: 10.1038/357167a0. [DOI] [PubMed] [Google Scholar]