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Volume 147, Issue 11

SvpA, a novel surface virulence-associated protein required for intracellular survival of Free

Abstract

A previously unknown protein, designated SvpA (urface irulence-associated rotein) and implicated in the virulence of the intracellular pathogen , was identified. This 64 kDa protein, encoded by , is both secreted in culture supernatants and surface-exposed, as shown by immunogold labelling of whole bacteria with an anti-SvpA antibody. Analysis of the peptide sequence revealed that SvpA contains a leader peptide, a predicted C-terminal transmembrane region and a positively charged tail resembling that of the surface protein ActA, suggesting that SvpA might partially reassociate with the bacterial surface by its C-terminal membrane anchor. An allelic mutant was constructed by disrupting in the wild-type strain LO28. The virulence of this mutant was strongly attenuated in the mouse, with a 2 log decrease in the LD and restricted bacterial growth in organs as compared to the wild-type strain. This reduced virulence was not related either to a loss of adherence or to a lower expression of known virulence factors, which remained unaffected in the mutant. It was caused by a restriction of intracellular growth of mutant bacteria. By following the intracellular behaviour of bacteria within bone-marrow-derived macrophages by confocal and electron microscopy studies, it was found that most mutant bacteria remained confined within phagosomes, in contrast to wild-type bacteria which rapidly escaped to the cytoplasm. The regulation of was independent of PrfA, the transcriptional activator of virulence genes in . In fact, SvpA was down-regulated by MecA, ClpC and ClpP, which are highly homologous to proteins of forming a regulatory complex controlling the competence state of this saprophyte. The results indicate that: (i) SvpA is a novel factor involved in the virulence of . , promoting bacterial escape from phagosomes of macrophages; (ii) SvpA is, at least partially, associated with the surface of bacteria; and (iii) SvpA is PrfA-independent and controlled by a MecA-dependent regulatory network.

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Keyword(s): bacterial competence , microbial pathogenicity and surface protein
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2001-11-01
2024-04-24
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References

  1. Borezée, E., Msadek, T., Durant, L. & Berche, P. (2000a). Identification in Listeria monocytogenes of MecA, a homologue of the Bacillus subtilis competence regulatory protein. J Bacteriol 182, 5931-5934.[CrossRef] [Google Scholar]
  2. Borezée, E., Pellegrini, E. & Berche, P. (2000b). OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect Immun 68, 7069-7077.[CrossRef] [Google Scholar]
  3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248-254.[CrossRef] [Google Scholar]
  4. Celli, J. & Trieu-Cuot, P. (1998). Circularization of Tn916 is required for expression of the transposon-encoded transfer functions: characterization of long tetracycline-inducible transcripts reading through the attachment site. Mol Microbiol 28, 103-117. [Google Scholar]
  5. Chakraborty, T. (1999). Molecular and cell biological aspects of infection by Listeria monocytogenes. Immunobiology 201, 155-163.[CrossRef] [Google Scholar]
  6. Chakraborty, T., Leimeister-Wachter, M., Domann, E., Hartl, M., Goebel, W., Nichterlein, T. & Notermans, S. (1992). Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol 174, 568-574. [Google Scholar]
  7. de Chastellier, C. & Berche, P. (1994). Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria. Infect Immun 62, 543-553. [Google Scholar]
  8. Cossart, P. & Lecuit, M. (1998). Interactions of Listeria monocytogenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling. EMBO J 17, 3797-3806.[CrossRef] [Google Scholar]
  9. Domann, E., Wehland, J., Rohde, M. & 7 other authors (1992). A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J 11, 1981–1990. [Google Scholar]
  10. Gaillard, J. L. & Finlay, B. B. (1996). Effect of cell polarization and differentiation on entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line. Infect Immun 64, 1299-1308. [Google Scholar]
  11. Gaillard, J. L., Berche, P., Mounier, J., Richard, S. & Sansonetti, P. (1987).In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun 55, 2822-2829. [Google Scholar]
  12. Gaillard, J. L., Berche, P., Frehel, C., Gouin, E. & Cossart, P. (1991). Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell 65, 1127-1141.[CrossRef] [Google Scholar]
  13. Gaillard, J. L., Jaubert, F. & Berche, P. (1996). The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. J Exp Med 183, 359-369.[CrossRef] [Google Scholar]
  14. Gaillot, O., Pellegrini, E., Bregenholt, S., Nair, S. & Berche, P. (2000). The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol Microbiol 35, 1286-1294. [Google Scholar]
  15. Geoffroy, C., Raveneau, J., Beretti, J. L., Lecroisey, A., Vazquez-Boland, J. A., Alouf, J. E. & Berche, P. (1991). Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes. Infect Immun 59, 2382-2388. [Google Scholar]
  16. Gottesman, S. & Maurizi, M. R. (1992). Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev 56, 592-621. [Google Scholar]
  17. Gottesman, S., Roche, E., Zhou, Y. & Sauer, R. T. (1998). The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev 12, 1338-1347.[CrossRef] [Google Scholar]
  18. Gray, M. L. & Killinger, A. H. (1966).Listeria monocytogenes and listeric infections. Bacteriol Rev 30, 309-382. [Google Scholar]
  19. Kay, B. K., Williamson, M. P. & Sudol, M. (2000). The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J 14, 231-241. [Google Scholar]
  20. Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H. & Cossart, P. (1992).L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68, 521-531.[CrossRef] [Google Scholar]
  21. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  22. Larsen, C. N. & Finley, D. (1997). Protein translocation channels in the proteasome and other proteases. Cell 91, 431-434.[CrossRef] [Google Scholar]
  23. Lebrun, M., Mengaud, J., Ohayon, H., Nato, F. & Cossart, P. (1996). Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells. Mol Microbiol 21, 579-592.[CrossRef] [Google Scholar]
  24. Menard, R., Sansonetti, P. J. & Parsot, C. (1993). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899-5906. [Google Scholar]
  25. Milohanic, E., Pron, B., Berche, P. & Gaillard, J. L. (2000). Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells. European Listeria Genome Consortium. Microbiology 146, 731-739. [Google Scholar]
  26. Msadek, T., Dartois, V., Kunst, F., Herbaud, M. L., Denizot, F. & Rapoport, G. (1998). ClpP of Bacillus subtilis is required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation. Mol Microbiol 27, 899-914.[CrossRef] [Google Scholar]
  27. 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]
  28. Niebuhr, K., Ebel, F., Frank, R. & 7 other authors (1997). A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J 16, 5433–5444.[CrossRef] [Google Scholar]
  29. Poyart, C., Abachin, E., Razafimanantsoa, I. & Berche, P. (1993). The zinc metalloprotease of Listeria monocytogenes is required for maturation of phosphatidylcholine phospholipase C: direct evidence obtained by gene complementation. Infect Immun 61, 1576-1580. [Google Scholar]
  30. 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]
  31. Rouquette, C., de Chastellier, C., Nair, S. & Berche, P. (1998). The ClpC ATPase 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]
  32. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  33. 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]
  34. Squires, C. & Squires, C. L. (1992). The Clp proteins: proteolysis regulators or molecular chaperones? J Bacteriol 174, 1081-1085. [Google Scholar]
  35. Tilney, L. G. & Portnoy, D. A. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J Cell Biol 109, 1597-1608.[CrossRef] [Google Scholar]
  36. Turgay, K., Hamoen, L. W., Venema, G. & Dubnau, D. (1997). Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of Bacillus subtilis. Genes Dev 11, 119-128.[CrossRef] [Google Scholar]
  37. Turgay, K., Hahn, J., Burghoorn, J. & Dubnau, D. (1998). Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J 17, 6730-6738.[CrossRef] [Google Scholar]
  38. Wang, J., Hartling, J. A. & Flanagan, J. M. (1997). The structure of ClpP at 2·3 Å resolution suggests a model for ATP-dependent proteolysis. Cell 91, 447-456.[CrossRef] [Google Scholar]
  39. 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]
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SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes
Microbiology 147, 2913 (2001); https://doi.org/10.1099/00221287-147-11-2913
/content/journal/micro/10.1099/00221287-147-11-2913
/content/journal/micro/10.1099/00221287-147-11-2913
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