Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Nov;177(22):6469–6476. doi: 10.1128/jb.177.22.6469-6476.1995

Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator.

B Sheehan 1, A Klarsfeld 1, T Msadek 1, P Cossart 1
PMCID: PMC177497  PMID: 7592422

Abstract

PrfA is a pleiotropic activator of virulence gene expression in the pathogenic bacterium Listeria monocytogenes. Several lines of evidence have suggested that a hierarchy of virulence gene activation by PrfA exists. This hypothesis was investigated by assessing the ability of PrfA to activate the expression of virulence gene fusions to lacZ in Bacillus subtilis. Expression of PrfA in this heterologous host was sufficient for activation of transcription at the hly, plcA, mpl, and actA promoters. Activation was most efficient at the divergently transcribed hly and plcA promoters. The putative PrfA binding site shared by these promoters is perfectly symmetrical and appears to represent the optimum sequence for target gene activation by PrfA. The activation of actA and mpl expression was considerably weaker and occurred more slowly than that observed at the hly and plcA promoters, suggesting that greater quantities of PrfA are required for productive interaction at these promoters. Interestingly, expression of an inlA-lacZ transcriptional fusion was very poorly activated by PrfA in B. subtilis, suggesting that other Listeria factors, in addition to PrfA, are required for PrfA-mediated activation at this promoter. Further support for the involvement of such factors was obtained by constructing and analyzing a prfA deletion mutant of L. monocytogenes. We observed that, in contrast to that of the other genes of the PrfA regulon, expression of inlA is only partially dependent on PrfA.

Full Text

The Full Text of this article is available as a PDF (342.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bohne J., Sokolovic Z., Goebel W. Transcriptional regulation of prfA and PrfA-regulated virulence genes in Listeria monocytogenes. Mol Microbiol. 1994 Mar;11(6):1141–1150. doi: 10.1111/j.1365-2958.1994.tb00390.x. [DOI] [PubMed] [Google Scholar]
  2. Brundage R. A., Smith G. A., Camilli A., Theriot J. A., Portnoy D. A. Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11890–11894. doi: 10.1073/pnas.90.24.11890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Camilli A., Tilney L. G., Portnoy D. A. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol. 1993 Apr;8(1):143–157. doi: 10.1111/j.1365-2958.1993.tb01211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chakraborty T., Leimeister-Wächter M., Domann E., Hartl M., Goebel W., Nichterlein T., Notermans S. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol. 1992 Jan;174(2):568–574. doi: 10.1128/jb.174.2.568-574.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Cossart P. Actin-based bacterial motility. Curr Opin Cell Biol. 1995 Feb;7(1):94–101. doi: 10.1016/0955-0674(95)80050-6. [DOI] [PubMed] [Google Scholar]
  7. Cossart P., Kocks C. The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes. Mol Microbiol. 1994 Aug;13(3):395–402. doi: 10.1111/j.1365-2958.1994.tb00434.x. [DOI] [PubMed] [Google Scholar]
  8. Cossart P., Mengaud J. Listeria monocytogenes. A model system for the molecular study of intracellular parasitism. Mol Biol Med. 1989 Oct;6(5):463–474. [PubMed] [Google Scholar]
  9. Cossart P., Vicente M. F., Mengaud J., Baquero F., Perez-Diaz J. C., Berche P. Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun. 1989 Nov;57(11):3629–3636. doi: 10.1128/iai.57.11.3629-3636.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Debarbouille M., Arnaud M., Fouet A., Klier A., Rapoport G. The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators. J Bacteriol. 1990 Jul;172(7):3966–3973. doi: 10.1128/jb.172.7.3966-3973.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DiRita V. J., Parsot C., Jander G., Mekalanos J. J. Regulatory cascade controls virulence in Vibrio cholerae. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5403–5407. doi: 10.1073/pnas.88.12.5403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Domann E., Leimeister-Wächter M., Goebel W., Chakraborty T. Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene. Infect Immun. 1991 Jan;59(1):65–72. doi: 10.1128/iai.59.1.65-72.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Domann E., Wehland J., Rohde M., Pistor S., Hartl M., Goebel W., Leimeister-Wächter M., Wuenscher M., Chakraborty T. 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. 1992 May;11(5):1981–1990. doi: 10.1002/j.1460-2075.1992.tb05252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dramsi S., Dehoux P., Cossart P. Common features of gram-positive bacterial proteins involved in cell recognition. Mol Microbiol. 1993 Sep;9(5):1119–1121. doi: 10.1111/j.1365-2958.1993.tb01241.x. [DOI] [PubMed] [Google Scholar]
  15. Dramsi S., Kocks C., Forestier C., Cossart P. Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator prfA. Mol Microbiol. 1993 Sep;9(5):931–941. doi: 10.1111/j.1365-2958.1993.tb01223.x. [DOI] [PubMed] [Google Scholar]
  16. Freitag N. E., Portnoy D. A. Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo. Mol Microbiol. 1994 Jun;12(5):845–853. doi: 10.1111/j.1365-2958.1994.tb01070.x. [DOI] [PubMed] [Google Scholar]
  17. Freitag N. E., Rong L., Portnoy D. A. Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread. Infect Immun. 1993 Jun;61(6):2537–2544. doi: 10.1128/iai.61.6.2537-2544.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Freitag N. E., Youngman P., Portnoy D. A. Transcriptional activation of the Listeria monocytogenes hemolysin gene in Bacillus subtilis. J Bacteriol. 1992 Feb;174(4):1293–1298. doi: 10.1128/jb.174.4.1293-1298.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gaillard J. L., Berche P., Frehel C., Gouin E., Cossart P. Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell. 1991 Jun 28;65(7):1127–1141. doi: 10.1016/0092-8674(91)90009-n. [DOI] [PubMed] [Google Scholar]
  20. Goldfine H., Johnston N. C., Knob C. Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes. J Bacteriol. 1993 Jul;175(14):4298–4306. doi: 10.1128/jb.175.14.4298-4306.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goldfine H., Knob C. Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. Infect Immun. 1992 Oct;60(10):4059–4067. doi: 10.1128/iai.60.10.4059-4067.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holm L., Sander C., Rüterjans H., Schnarr M., Fogh R., Boelens R., Kaptein R. LexA repressor and iron uptake regulator from Escherichia coli: new members of the CAP-like DNA binding domain superfamily. Protein Eng. 1994 Dec;7(12):1449–1453. doi: 10.1093/protein/7.12.1449. [DOI] [PubMed] [Google Scholar]
  23. Huh Y. J., Weiss A. A. A 23-kilodalton protein, distinct from BvgA, expressed by virulent Bordetella pertussis binds to the promoter region of vir-regulated toxin genes. Infect Immun. 1991 Jul;59(7):2389–2395. doi: 10.1128/iai.59.7.2389-2395.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Janzon L., Arvidson S. The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. EMBO J. 1990 May;9(5):1391–1399. doi: 10.1002/j.1460-2075.1990.tb08254.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Klarsfeld A. D., Goossens P. L., Cossart P. Five Listeria monocytogenes genes preferentially expressed in infected mammalian cells: plcA, purH, purD, pyrE and an arginine ABC transporter gene, arpJ. Mol Microbiol. 1994 Aug;13(4):585–597. doi: 10.1111/j.1365-2958.1994.tb00453.x. [DOI] [PubMed] [Google Scholar]
  26. Kocks C., Gouin E., Tabouret M., Berche P., Ohayon H., Cossart P. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell. 1992 Feb 7;68(3):521–531. doi: 10.1016/0092-8674(92)90188-i. [DOI] [PubMed] [Google Scholar]
  27. Kunst F., Rapoport G. Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol. 1995 May;177(9):2403–2407. doi: 10.1128/jb.177.9.2403-2407.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lampidis R., Gross R., Sokolovic Z., Goebel W., Kreft J. The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators. Mol Microbiol. 1994 Jul;13(1):141–151. doi: 10.1111/j.1365-2958.1994.tb00409.x. [DOI] [PubMed] [Google Scholar]
  29. Leimeister-Wächter M., Domann E., Chakraborty T. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J Bacteriol. 1992 Feb;174(3):947–952. doi: 10.1128/jb.174.3.947-952.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Leimeister-Wächter M., Haffner C., Domann E., Goebel W., Chakraborty T. Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8336–8340. doi: 10.1073/pnas.87.21.8336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Maguin E., Duwat P., Hege T., Ehrlich D., Gruss A. New thermosensitive plasmid for gram-positive bacteria. J Bacteriol. 1992 Sep;174(17):5633–5638. doi: 10.1128/jb.174.17.5633-5638.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mengaud J., Dramsi S., Gouin E., Vazquez-Boland J. A., Milon G., Cossart P. Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol Microbiol. 1991 Sep;5(9):2273–2283. doi: 10.1111/j.1365-2958.1991.tb02158.x. [DOI] [PubMed] [Google Scholar]
  33. Mengaud J., Geoffroy C., Cossart P. Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases. Infect Immun. 1991 Mar;59(3):1043–1049. doi: 10.1128/iai.59.3.1043-1049.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mengaud J., Vicente M. F., Cossart P. Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation. Infect Immun. 1989 Dec;57(12):3695–3701. doi: 10.1128/iai.57.12.3695-3701.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Michel E., Reich K. A., Favier R., Berche P., Cossart P. Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O. Mol Microbiol. 1990 Dec;4(12):2167–2178. doi: 10.1111/j.1365-2958.1990.tb00578.x. [DOI] [PubMed] [Google Scholar]
  36. Miller J. F., Roy C. R., Falkow S. Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli. J Bacteriol. 1989 Nov;171(11):6345–6348. doi: 10.1128/jb.171.11.6345-6348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Park S. F., Kroll R. G. Expression of listeriolysin and phosphatidylinositol-specific phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes. Mol Microbiol. 1993 May;8(4):653–661. doi: 10.1111/j.1365-2958.1993.tb01609.x. [DOI] [PubMed] [Google Scholar]
  38. Portnoy D. A., Chakraborty T., Goebel W., Cossart P. Molecular determinants of Listeria monocytogenes pathogenesis. Infect Immun. 1992 Apr;60(4):1263–1267. doi: 10.1128/iai.60.4.1263-1267.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sheehan B., Kocks C., Dramsi S., Gouin E., Klarsfeld A. D., Mengaud J., Cossart P. Molecular and genetic determinants of the Listeria monocytogenes infectious process. Curr Top Microbiol Immunol. 1994;192:187–216. doi: 10.1007/978-3-642-78624-2_9. [DOI] [PubMed] [Google Scholar]
  40. Sullivan M. A., Yasbin R. E., Young F. E. New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene. 1984 Jul-Aug;29(1-2):21–26. doi: 10.1016/0378-1119(84)90161-6. [DOI] [PubMed] [Google Scholar]
  41. Tobe T., Nagai S., Okada N., Adler B., Yoshikawa M., Sasakawa C. Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid. Mol Microbiol. 1991 Apr;5(4):887–893. doi: 10.1111/j.1365-2958.1991.tb00762.x. [DOI] [PubMed] [Google Scholar]
  42. Vazquez-Boland J. A., Kocks C., Dramsi S., Ohayon H., Geoffroy C., Mengaud J., Cossart P. Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun. 1992 Jan;60(1):219–230. doi: 10.1128/iai.60.1.219-230.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Weinrauch Y., Msadek T., Kunst F., Dubnau D. Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis. J Bacteriol. 1991 Sep;173(18):5685–5693. doi: 10.1128/jb.173.18.5685-5693.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. de Souza M. T., Lecadet M. M., Lereclus D. Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription. J Bacteriol. 1993 May;175(10):2952–2960. doi: 10.1128/jb.175.10.2952-2960.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES