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
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-5-1439
2002-05-01
2019-08-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/5/1481439a.html?itemId=/content/journal/micro/10.1099/00221287-148-5-1439&mimeType=html&fmt=ahah

References

  1. Acha, P. & Szylres, B. (1980). Zoonoses and Communicable Diseases Common to Man and Animals. Washington, DC: Pan American Health Organization.
  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., van der Goot, G., Sola-Landa, A., Lopez-Goni, I., Moreno, E. & Gorvel, J. P. ( 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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-5-1439
Loading
/content/journal/micro/10.1099/00221287-148-5-1439
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error