1887

Abstract

Some rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a inactivation mutant (BMΔvirB) and an overexpression strain (BM-VIR) of a smooth strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMΔvirB. The quorum sensing signal molecule -dodecanoyl--homoserine lactone (C-HSL), a molecule that can inhibit expression of , inhibited the cytotoxicity of BM and BM-VIR, but not of BMΔvirB. These results indicated that overexpression of is responsible for cytotoxicity in macrophages. Transcription analysis showed that is regulated in a cell-density-dependent manner both in culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of are detrimental for intracellular survival also indicated that the expression of is tightly regulated in a cell-density-dependent manner.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.030619-0
2009-10-01
2020-01-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/10/3392.html?itemId=/content/journal/micro/10.1099/mic.0.030619-0&mimeType=html&fmt=ahah

References

  1. Alvarez-Martinez, M. T., Machold, J., Weise, C., Schmidt-Eisenlohr, H., Baron, C. & Rouot, B. ( 2001; ). The Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon chvE is essential for sugar utilization but not for survival in macrophages. J Bacteriol 183, 5343–5351.[CrossRef]
    [Google Scholar]
  2. Boschiroli, M. L., Ouahrani-Bettache, S., Foulongne, V., Michaux-Charachon, S., Bourg, G., Allardet-Servent, A., Cazevieille, C., Lavigne, J. P., Liautard, J. P. & other authors ( 2002a; ). Type IV secretion and Brucella virulence. Vet Microbiol 90, 341–348.[CrossRef]
    [Google Scholar]
  3. Boschiroli, M. L., Ouahrani-Bettache, S., Foulongne, V., Michaux-Charachon, S., Bourg, G., Allardet-Servent, A., Cazevieille, C., Liautard, J. P., Ramuz, M. & O'Callaghan, D. ( 2002b; ). The Brucella suis virB operon is induced intracellularly in macrophages. Proc Natl Acad Sci U S A 99, 1544–1549.[CrossRef]
    [Google Scholar]
  4. Cascales, E. & Christie, P. J. ( 2003; ). The versatile bacterial type IV secretion systems. Nat Rev Microbiol 1, 137–149.[CrossRef]
    [Google Scholar]
  5. Celli, J., de Chastellier, C., Franchini, D. M., Pizarro-Cerda, J., Moreno, E. & Gorvel, J. P. ( 2003; ). Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J Exp Med 198, 545–556.[CrossRef]
    [Google Scholar]
  6. Celli, J., Salcedo, S. P. & Gorvel, J. P. ( 2005; ). Brucella coopts the small GTPase Sar1 for intracellular replication. Proc Natl Acad Sci U S A 102, 1673–1678.[CrossRef]
    [Google Scholar]
  7. Comerci, D. J., Martinez-Lorenzo, M. J., Sieira, R., Gorvel, J. P. & 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]
  8. de Jong, M. F., Sun, Y. H., den Hartigh, A. B., van Dijl, J. M. & Tsolis, R. M. ( 2008; ). Identification of VceA and VceC, two members of the VjbR regulon that are translocated into macrophages by the Brucella type IV secretion system. Mol Microbiol 70, 1378–1396.[CrossRef]
    [Google Scholar]
  9. Delrue, R. M., Deschamps, C., Leonard, S., Nijskens, C., Danese, I., Schaus, J. M., Bonnot, S., Ferooz, J., Tibor, A. & other authors ( 2005; ). A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis. Cell Microbiol 7, 1151–1161.[CrossRef]
    [Google Scholar]
  10. Foulongne, V., Bourg, G., Cazevieille, C., Michaux-Charachon, S. & O'Callaghan, D. ( 2000; ). Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis. Infect Immun 68, 1297–1303.[CrossRef]
    [Google Scholar]
  11. Freeman, B. A. & Rumack, B. H. ( 1964; ). Cytopathogenic effect of Brucella spheroplasts on monocytes in tissue culture. J Bacteriol 88, 1310–1315.
    [Google Scholar]
  12. Gorvel, J. P. & Moreno, E. ( 2002; ). Brucella intracellular life: from invasion to intracellular replication. Vet Microbiol 90, 281–297.[CrossRef]
    [Google Scholar]
  13. Gross, A., Terraza, A., Ouahrani-Bettache, S., Liautard, J. P. & Dornand, J. ( 2000; ). In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells. Infect Immun 68, 342–351.[CrossRef]
    [Google Scholar]
  14. Hong, P. C., Tsolis, R. M. & Ficht, T. A. ( 2000; ). Identification of genes required for chronic persistence of Brucella abortus in mice. Infect Immun 68, 4102–4107.[CrossRef]
    [Google Scholar]
  15. Jimenez de Bagues, M. P., Terraza, A., Gross, A. & Dornand, J. ( 2004; ). Different responses of macrophages to smooth and rough Brucella spp.: relationship to virulence. Infect Immun 72, 2429–2433.[CrossRef]
    [Google Scholar]
  16. Kim, S., Watarai, M., Makino, S. & Shirahata, T. ( 2002; ). Membrane sorting during swimming internalization of Brucella is required for phagosome trafficking decisions. Microb Pathog 33, 225–237.[CrossRef]
    [Google Scholar]
  17. 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]
  18. Lestrate, P., Delrue, R. M., Danese, I., Didembourg, C., Taminiau, B., Mertens, P., De Bolle, X., Tibor, A., Tang, C. M. & Letesson, J. J. ( 2000; ). Identification and characterization of in vivo attenuated mutants of Brucella melitensis. Mol Microbiol 38, 543–551.[CrossRef]
    [Google Scholar]
  19. Livak, K. J. & Schmittgen, T. D. ( 2001; ). Analysis of relative gene expression data using real-time quantitative PCR and the method. Methods 25, 402–408.[CrossRef]
    [Google Scholar]
  20. Mota, L. J., Journet, L., Sorg, I., Agrain, C. & Cornelis, G. R. ( 2005; ). Bacterial injectisomes: needle length does matter. Science 307, 1278 [CrossRef]
    [Google Scholar]
  21. Nijskens, C., Copin, R., De Bolle, X. & Letesson, J. J. ( 2008; ). Intracellular rescuing of a B. melitensis 16M virB mutant by co-infection with a wild type strain. Microb Pathog 45, 134–141.[CrossRef]
    [Google Scholar]
  22. 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]
  23. Pei, J. & Ficht, T. A. ( 2004; ). Brucella abortus rough mutants are cytopathic for macrophages in culture. Infect Immun 72, 440–450.[CrossRef]
    [Google Scholar]
  24. Pei, J., Turse, J. E., Wu, Q. & Ficht, T. A. ( 2006; ). Brucella abortus rough mutants induce macrophage oncosis that requires bacterial protein synthesis and direct interaction with the macrophage. Infect Immun 74, 2667–2675.[CrossRef]
    [Google Scholar]
  25. Pei, J., Wu, Q., Kahl-McDonagh, M. & Ficht, T. A. ( 2008; ). Cytotoxicity in macrophages infected with rough Brucella mutants is type IV secretion system dependent. Infect Immun 76, 30–37.[CrossRef]
    [Google Scholar]
  26. Pizarro-Cerda, J., Meresse, S., Parton, R. G., van der Goot, G., Sola-Landa, A., Lopez-Goni, I., Moreno, E. & Gorvel, J. P. ( 1998; ). Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect Immun 66, 5711–5724.
    [Google Scholar]
  27. Sieira, R., Comerci, D. J., Sanchez, D. O. & Ugalde, R. A. ( 2000; ). A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 182, 4849–4855.[CrossRef]
    [Google Scholar]
  28. Starr, T., Ng, T. W., Wehrly, T. D., Knodler, L. A. & Celli, J. ( 2008; ). Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment. Traffic 9, 678–694.[CrossRef]
    [Google Scholar]
  29. Taminiau, B., Daykin, M., Swift, S., Boschiroli, M. L., Tibor, A., Lestrate, P., De Bolle, X., O'Callaghan, D., Williams, P. & Letesson, J. J. ( 2002; ). Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis. Infect Immun 70, 3004–3011.[CrossRef]
    [Google Scholar]
  30. Teixeira-Gomes, A. P., Cloeckaert, A. & Zygmunt, M. S. ( 2000; ). Characterization of heat, oxidative, and acid stress responses in Brucella melitensis. Infect Immun 68, 2954–2961.[CrossRef]
    [Google Scholar]
  31. Wang, Y., Chen, Z., Qiao, F., Ying, T., Yuan, J., Zhong, Z., Zhou, L., Du, X., Wang, Z. & other authors ( 2009; ). Comparative proteomics analyses reveal the virB of B. melitensis affects expression of intracellular survival related proteins. PLoS One 4, e5368 [CrossRef]
    [Google Scholar]
  32. Watarai, M., Makino, S., Fujii, Y., Okamoto, K. & Shirahata, T. ( 2002; ). Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cell Microbiol 4, 341–355.[CrossRef]
    [Google Scholar]
  33. West, N. P., Sansonetti, P., Mounier, J., Exley, R. M., Parsot, C., Guadagnini, S., Prévost, M. C., Prochnicka-Chalufour, A., Delepierre, M. & other authors ( 2005; ). Optimization of virulence functions through glucosylation of Shigella LPS. Science 307, 1313–1317.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.030619-0
Loading
/content/journal/micro/10.1099/mic.0.030619-0
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