1887

Abstract

The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones. In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen , which showed 70% identity to ClpA of , was identified and sequenced. Following heterologous expression in strains SG1126 (Δ) and SG1127 (Δ Δ), b-ClpA replaced the function of ClpA, participating in the degradation of abnormal proteins. A null mutant of was constructed, and growth experiments at 37 and 42 °C showed reduced growth rates for the null mutant, especially at the elevated temperature. The mutant complemented by and overexpressing the gene was even more impaired at 37 and 42 °C. In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the null mutant and, to a lesser extent, the strain of overexpressing behaved similarly to the wild-type strain. In a murine model of infection, however, the absence of ClpA significantly increased persistence of . These results showed that in the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.

Keyword(s): Brucella , chaperone , ClpA and ClpATPase
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2000-07-01
2019-09-24
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References

  1. Allen, C. A., Adams, L. G. & Ficht, T. A. ( 1998; ). Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival. Infect Immun 66, 1008-1016.
    [Google Scholar]
  2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403-410.[CrossRef]
    [Google Scholar]
  3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seichman, J. G., Smith, J. A. & Struhl, K. (1989). Current Protocols in Molecular Biology. New York: Wiley.
  4. Bairoch, A., Bucher, P. & Hofmann, K. ( 1997; ). The PROSITE database, its status in 1997. Nucleic Acids Res 25, 217-221.[CrossRef]
    [Google Scholar]
  5. Blum, P., Ory, J., Bauernfeind, J. & Krska, J. ( 1992; ). Physiological consequences of DnaK and DnaJ overproduction in Escherichia coli. J Bacteriol 174, 7436-7444.
    [Google Scholar]
  6. Bosseray, N. & Plommet, M. ( 1976; ). Transformation normalisant la distribution du nombre de Brucella dans la rate de souris inoculées par voie intrapéritoneale. J Biol Stand 4, 341-351.[CrossRef]
    [Google Scholar]
  7. Bosseray, N. & Plommet, M. ( 1990; ). Brucella suis S2, Brucella melitensis Rev. 1 and Brucella abortus S19 living vaccines: residual virulence and immunity induced against three Brucella species challenge strains in mice. Vaccine 8, 462-468.[CrossRef]
    [Google Scholar]
  8. Caron, E., Liautard, J. P. & Köhler, S. ( 1994; ). Differentiated U937 cells exhibit increased bactericidal activity upon LPS activation and discriminate between virulent and avirulent Listeria and Brucella species. J Leukoc Biol 56, 174-181.
    [Google Scholar]
  9. Corbel, M. J. ( 1990; ). Brucella. In Principles of Bacteriology, Virology and Immunity, pp. 339-353. Edited by M. T. Parker & L. H. Collier. London: Edward Arnold.
  10. Donnenberg, M. S. & Kaper, J. B. ( 1991; ). Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59, 4310-4317.
    [Google Scholar]
  11. Drazek, E. S., Houng, H.-S. H., Crawford, R. M., Hadfield, T. L., Hoover, D. L. & Warren, R. L. ( 1995; ). Deletion of purE attenuates Brucella melitensis 16M for growth in human monocyte-derived macrophages. Infect Immun 63, 3297-3301.
    [Google Scholar]
  12. Elzer, P. H., Phillips, R. W., Kovach, M. E., Peterson, K. M. & Roop, R. M.II ( 1994; ). Characterization and genetic complementation of a Brucella abortus high-temperature requirement A (htrA) deletion mutant. Infect Immun 62, 4135-4139.
    [Google Scholar]
  13. Gay, P., Le Coq, D., Steinmetz, M., Ferrari, E. & Hoch, J. A. ( 1983; ). Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli. J Bacteriol 153, 1424-1431.
    [Google Scholar]
  14. Gay, P., Le Coq, D., Steinmetz, M., Berkelman, T. & Kado, C. I. ( 1985; ). Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol 164, 918-921.
    [Google Scholar]
  15. Godfroid, F., Taminiau, B., Danese, I., Denoel, P., Tibor, A., Weynants, V., Cloeckaert, A., Godfroid, J. & Letesson, J. J. ( 1998; ). Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages. Infect Immun 66, 5485-5493.
    [Google Scholar]
  16. Gottesman, S., Squires, C., Pichersky, E. & 11 other authors ( 1990; a). Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes. Proc Natl Acad Sci USA 87, 3513–3517.[CrossRef]
    [Google Scholar]
  17. Gottesman, S., Clark, W. P. & Maurizi, M. R. ( 1990b; ). The ATP-dependent Clp protease of Escherichia coli. J Biol Chem 265, 7886-7893.
    [Google Scholar]
  18. Gottesman, S., Clark, W. P., de Crecy-Lagard, V. & Maurizi, M. R. ( 1993; ). ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. J Biol Chem 268, 22618-22626.
    [Google Scholar]
  19. Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  20. Harmon, B. G., Adams, L. G. & Frey, M. ( 1988; ). Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages. Am J Vet Res 49, 1092-1097.
    [Google Scholar]
  21. Hendrick, J. P. & Hartl, F. U. ( 1993; ). Molecular chaperone function of heat-shock proteins. Annu Rev Biochem 62, 349-384.[CrossRef]
    [Google Scholar]
  22. Hoskins, J. R., Pak, M., Maurizi, M. R. & Wickner, S. ( 1998; ). The role of the ClpA chaperone in proteolysis by ClpAP. Proc Natl Acad Sci USA 95, 12135-12140.[CrossRef]
    [Google Scholar]
  23. Hwang, B. J., Woo, K. M., Goldberg, A. L. & Chung, C. H. ( 1988; ). Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits. J Biol Chem 263, 8727-8734.
    [Google Scholar]
  24. Jenal, U. & Fuchs, T. ( 1998; ). An essential protease involved in bacterial cell-cycle control. EMBO J 17, 5658-5669.[CrossRef]
    [Google Scholar]
  25. Katayama, Y., Gottesman, S., Pumphrey, J., Rudikoff, S., Clark, W. P. & Maurizi, M. R. ( 1988; ). The two-component, ATP-dependent Clp protease of Escherichia coli: purification, cloning, and mutational analysis of the ATP-binding component. J Biol Chem 263, 15226-15236.
    [Google Scholar]
  26. Köhler, S., Teyssier, J., Cloeckaert, A., Rouot, B. & Liautard, J. P. ( 1996; ). Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937-derived phagocytes. Mol Microbiol 20, 701-712.[CrossRef]
    [Google Scholar]
  27. Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M.II & Peterson, K. M. ( 1994; ). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 800-802.
    [Google Scholar]
  28. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  29. Liautard, J. P., Gross, A., Dornand, J. & Köhler, S. ( 1996; ). Interactions between professional phagocytes and Brucella spp. Microbiologia 12, 197-206.
    [Google Scholar]
  30. Lin, J. & Ficht, T. A. ( 1995; ). Protein synthesis in Brucella abortus induced during macrophage infection. Infect Immun 63, 1409-1414.
    [Google Scholar]
  31. Lin, J., Adams, L. G. & Ficht, T. A. ( 1992; ). Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins. Infect Immun 60, 2425-2431.
    [Google Scholar]
  32. Maurizi, M. R., Clark, W. P., Katayama, Y., Rudikoff, S., Pumphrey, J., Bowers, B. & Gottesman, S. ( 1990; ). Sequence and structure of ClpP, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J Biol Chem 265, 12536-12545.
    [Google Scholar]
  33. Nair, S., Frehel, C., Nguyen, L., Escuyer, V. & Berche, P. ( 1999; ). ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes. Mol Microbiol 31, 185-196.[CrossRef]
    [Google Scholar]
  34. O’Callaghan, D., Cazevieille, C., Allardet-Servent, A., Boschiroli, M. L., Bourg, G., Foulongne, V., Frutos, P., Kulakov, Y. & Ramuz, M. ( 1999; ). A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis. Mol Microbiol 33, 1210-1220.
    [Google Scholar]
  35. Pak, M. & Wickner, S. ( 1997; ). Mechanism of protein remodeling by ClpA chaperone. Proc Natl Acad Sci USA 94, 4901-4906.[CrossRef]
    [Google Scholar]
  36. Pearson, W. R. & Lipman, D. J. ( 1988; ). Improved tools for biological sequence analysis. Proc Natl Acad Sci USA 85, 2444-2448.[CrossRef]
    [Google Scholar]
  37. Porte, F., Liautard, J. P. & Köhler, S. ( 1999; ). Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages. Infect Immun 67, 4041-4047.
    [Google Scholar]
  38. Price, R. E., Templeton, J. W., Smith, R.III & Adams, L. G. ( 1990; ). Ability of mononuclear phagocytes from cattle naturally resistant or susceptible to brucellosis to control in vitro intracellular survival of Brucella abortus. Infect Immun 58, 879-886.
    [Google Scholar]
  39. Rafie-Kolpin, M., Essenberg, R. C. & Wyckoff, J. H.III ( 1996; ). Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus. Infect Immun 64, 5274-5283.
    [Google Scholar]
  40. Rost, B., Sander, C. & Schneider, R. ( 1994; ). PHD – an automatic mail server for protein secondary structure prediction. Comput Appl Biosci 10, 53-60.
    [Google Scholar]
  41. Rouquette, C., Ripio, M.-T., Pellegrini, E., Bolla, J.-M., Tascon, R. I., Vazquez-Boland, J.-A. & Berche, P. ( 1996; ). Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes. Mol Microbiol 21, 977-987.[CrossRef]
    [Google Scholar]
  42. Rouquette, C., de Chastellier, C., Nair, S. & Berche, P. ( 1998; ). The ClpATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages. Mol Microbiol 27, 1235-1245.[CrossRef]
    [Google Scholar]
  43. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  44. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef]
    [Google Scholar]
  45. Schirmer, E. C., Glover, J. R., Singer, M. A. & Lindquist, S. ( 1996; ). HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem Sci 21, 289-296.[CrossRef]
    [Google Scholar]
  46. Senior, A. E. ( 1988; ). ATP synthesis by oxidative phosphorylation. Physiol Rev 68, 177-231.
    [Google Scholar]
  47. Smith, R.III ( 1990; ). T lymphocyte-mediated mechanisms of acquired protective immunity against brucellosis in cattle. In Advances in Brucellosis Research, pp. 164-190. Edited by L. G. Adams. College Station, TX: Texas A&M University Press.
  48. Smith, C. K., Baker, T. A. & Sauer, R. T. ( 1999; ). Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc Natl Acad Sci USA 96, 6678-6682.[CrossRef]
    [Google Scholar]
  49. Sola-Landa, A., Pizarro-Cerda, J., Grillo, M.-J., Moreno, E., Moriyon, I., Blasco, J.-M., Gorvel, J. P. & Lopez-Goni, I. ( 1998; ). A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence. Mol Microbiol 29, 125-138.[CrossRef]
    [Google Scholar]
  50. Squires, C. & Squires, C. L. ( 1992; ). The Clp proteins: proteolysis regulators or molecular chaperones? J Bacteriol 174, 1081-1085.
    [Google Scholar]
  51. Suzuki, C. K., Rep, M., van Dijl, J. M., Suda, K., Grivell, L. A. & Schatz, G. ( 1997; ). ATP-dependent proteases that also chaperone protein biogenesis. Trends Biochem Sci 22, 118-123.[CrossRef]
    [Google Scholar]
  52. Thomas, J. G. & Baneyx, F. ( 1998; ). Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG in vivo. J Bacteriol 180, 5165-5172.
    [Google Scholar]
  53. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ( 1994; ). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[CrossRef]
    [Google Scholar]
  54. Tybulewicz, V. L., Falk, G. & Walker, J. E. ( 1984; ). Rhodopseudomonas blastica atp operon: nucleotide sequence and transcription. J Mol Biol 179, 185-214.[CrossRef]
    [Google Scholar]
  55. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. ( 1982; ). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1, 945-951.
    [Google Scholar]
  56. Wawrzynow, A., Banecki, B. & Zylicz, M. ( 1996; ). The Clp ATPases define a novel class of molecular chaperones. Mol Microbiol 21, 895-899.[CrossRef]
    [Google Scholar]
  57. Wickner, S., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K. & Maurizi, M. R. ( 1994; ). A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Natl Acad Sci USA 91, 12218-12222.[CrossRef]
    [Google Scholar]
  58. Woo, K. M., Chung, W. J., Ha, D. B., Goldberg, A. L. & Chung, C. H. ( 1989; ). Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides. J Biol Chem 264, 2088-2091.
    [Google Scholar]
  59. Young, E. ( 1983; ). Human brucellosis. Rev Infect Dis 5, 821-842.[CrossRef]
    [Google Scholar]
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