1887

Abstract

is a facultative intracellular bacterium capable of surviving inside macrophages. The VirB complex, which is highly similar to conjugative DNA transfer apparatuses, is required for intracellular replication. A conserved NTP-binding domain in VirB4 suggests that one or both proteins couple energy by NTP hydrolysis to transport of putative effector molecule(s). Here it is shown that a mutant strain of that contains an in-frame deletion in is unable to replicate in macrophages and survives in mice. Intracellular replication and virulence in mice are fully restored by expressing , indicating that VirB4 is essential for intracellular replication and virulence in mice. An alteration within the NTP-binding region of VirB4 by site-directed mutagenesis abolished complementation of a mutant, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Intracellular replication was inhibited in wild-type after introducing a plasmid expressing a mutant VirB4 altered in the NTP-binding region. The dominant negative phenotype suggests that VirB4 either functions as a multimer or interacts with some other component(s) necessary for intracellular replication. Wild-type -containing phagosomes lack the glycoprotein LAMP-1, which is an indicator of the normal endocytic pathway. Mutant strains were found in phagosomes that co-localized with LAMP-1, indicating that VirB4 containing the intact NTP-binding region is essential for evasion of fusion with lysosomes.

Keyword(s): macrophage and type IV secretion
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2002-05-01
2024-03-29
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References

  1. Acha P., Szylres B. 1980 Zoonoses and Communicable Diseases Common to Man and Animals Washington, DC: Pan American Health Organization;
    [Google Scholar]
  2. Andrews H. L., Vogel J. P., Isberg R. R. 1998; Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect Immun 66:950–958
    [Google Scholar]
  3. Arenas G. N., Staskevich A. S., Aballay A., Mayorga L. S. 2000; Intracellular trafficking of Brucella abortus in J774 macrophages. Infect Immun 68:4255–4263 [CrossRef]
    [Google Scholar]
  4. Baldwin C. L., Winter A. J. 1994; Macrophages and Brucella . Immunol Ser 60:363–380
    [Google Scholar]
  5. Berger B. R., Christie P. J. 1993; The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J Bacteriol 175:1723–1734
    [Google Scholar]
  6. Chen J. W., Cha Y., Yuksel K. U., Gracy R. W., August J. T. 1988; Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. J Biol Chem 263:8754–8758
    [Google Scholar]
  7. Christie P. J. 2001; Type IV secretion: intercellular transfer of macromolecules by systems ancestrally related to conjugation machines. Mol Microbiol 40:294–305 [CrossRef]
    [Google Scholar]
  8. Christie P. J., Vogel J. P. 2000; Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8:354–360 [CrossRef]
    [Google Scholar]
  9. Comerci D. J., Martinez-Lorenzo M. J., Sieira R., Gorvel J., Ugalde R. A. 2001; Essential role of the VirB machinery in the maturation of the Brucella abortus -containing vacuole. Cell Microbiol 3:159–168 [CrossRef]
    [Google Scholar]
  10. Cook D. M., Farizo K. M., Burns D. L. 1999; Identification and characterization of PtlC, an essential component of the pertussis toxin secretion system. Infect Immun 67:754–759
    [Google Scholar]
  11. Covacci A., Telford J. L., Del Giudice G., Parsonnet J., Rappuoli R. 1999; Helicobacter pylori virulence and genetic geography. Science 284:1328–1333 [CrossRef]
    [Google Scholar]
  12. Dang T. A., Zhou X. R., Graf B., Christie P. J. 1999; Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter. Mol Microbiol 32:1239–1253 [CrossRef]
    [Google Scholar]
  13. Harter C., Mellman I. 1992; Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol 177:311–325
    [Google Scholar]
  14. Kolter R., Inuzuka M., Helinski D. R. 1978; Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15:1199–1208 [CrossRef]
    [Google Scholar]
  15. Kotob S. I., Hausman S. Z., Burns D. L. 1995; Localization of the promoter for the ptl genes of Bordetella pertussis , which encode proteins essential for secretion of pertussis toxin. Infect Immun 63:3227–3230
    [Google Scholar]
  16. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Roop R. M.II., Peterson K. M. 1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [CrossRef]
    [Google Scholar]
  17. Kuldau G. A., De Vos G., Owen J., McCaffrey G., Zambryski P. 1990; The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Mol Gen Genet 221:256–266 [CrossRef]
    [Google Scholar]
  18. Liautard J. P., Gross A., Dornand J., Kohler S. 1996; Interactions between professional phagocytes and Brucella spp. Microbiologia 12:197–206
    [Google Scholar]
  19. McLean I. W., Nakane P. K. 1974; Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 22:1077–1083 [CrossRef]
    [Google Scholar]
  20. 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]
  21. Pizarro-Cerda J., Meresse S., Parton R. G., Sola-Landa A., Lopez-Goni I., Moreno E., Gorvel J. P., van der Goot G. 1998a; Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect Immun 66:5711–5724
    [Google Scholar]
  22. Pizarro-Cerda J., Moreno E., Sanguedolce V., Mege J. L., Gorvel J. P. 1998b; Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun 66:2387–2392
    [Google Scholar]
  23. Pohlman R. F., Genetti H. D., Winans S. C. 1994; Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol 14:655–668 [CrossRef]
    [Google Scholar]
  24. Sangari F. J., Aguero J. 1996; Molecular basis of Brucella pathogenicity: an update. Microbiologia 12:207–218
    [Google Scholar]
  25. Segal G., Purcell M., Shuman H. A. 1998; Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci U S A 95:1669–1674 [CrossRef]
    [Google Scholar]
  26. Sieira R., Comerci D. J., Sanchez D. O., Ugalde R. A. 2000; A homologue of an operon required for DNA transfer in Agrobacterium tumefaciens is required in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 182:4849–4855 [CrossRef]
    [Google Scholar]
  27. Sinai A. P., Joiner K. A. 1997; Safe haven: the cell biology of nonfusogenic pathogen vacuoles. Annu Rev Microbiol 51:415–462 [CrossRef]
    [Google Scholar]
  28. Stachel S. E., Nester E. W. 1986; The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens . EMBO J 5:1445–1454
    [Google Scholar]
  29. Swanson M. S., Isberg R. R. 1996; Identification of Legionella pneumophila mutants that have aberrant intracellular fates. Infect Immun 64:2585–2594
    [Google Scholar]
  30. Vogel J. P., Andrews H. L., Wong S. K., Isberg R. R. 1998; Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873–876 [CrossRef]
    [Google Scholar]
  31. Walker M. 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]
  32. Watarai M., Andrews H. L., Isberg R. R. 2001; Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth. Mol Microbiol 39:313–329 [CrossRef]
    [Google Scholar]
  33. Weiss A. A., Johnson F. D., Burns D. L. 1993; Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A 90:2970–2974 [CrossRef]
    [Google Scholar]
  34. Winans S. C., Walker G. C. 1985; Conjugal transfer system of the IncN plasmid pKM101. J Bacteriol 161:402–410
    [Google Scholar]
  35. Woodcock D. M., Crowther P. J., Doherty J., Jefferson S., DeCruz E., Noyer-Weidner M., Smith S. S., Michael M. Z., Graham M. W. 1989; Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17:3469–3478 [CrossRef]
    [Google Scholar]
  36. Zavala I., Nava A., Guerra J., Quiros C. 1994; Brucellosis. Infect Dis Clin N Am 8:225–241
    [Google Scholar]
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