Multiple point mutations in virulence genes explain the low virulence of field strains Free

Abstract

In order to understand the causes of the low virulence of field strains, five low-virulence strains were analysed. These five strains showed changes in relation to invasion, phosphatidyl-inositol phospholipase C (PI-PLC) activity, plaque formation and virulence. Molecular analyses revealed the same mutations in the , and genes in all five strains. The Thr262Ala substitution in the PI-PLC protein was responsible for the absence of PI-PLC activity. This residue, conserved in certain species, is located at the outer rim of the active site pocket and could impair the cleavage activity of the enzyme. The low invasion rate of these strains was due to a nonsense codon leading to a lack of InlA protein synthesis, and to an Ala117Thr substitution in the leucine-rich repeat of InlB, which altered the interaction with the Met receptor. Single complementation with the , or genes restored the capacity of low-virulence strains either to enter epithelial and fibroblastic cells or to express PI-PLC activity. Complementation by allelic exchange of the gene on the chromosome and complementation with either the or the gene restored the ability to form plaques, but only partly restored the virulence, suggesting that there were other gene mutation(s) with consequences that could mainly be observed . These results indicate that the low virulence of strains can be explained by point mutations in a number of virulence genes; these could therefore be important for detecting low-virulence strains. Moreover, the fact that all the strains had the same substitutions suggests that they have a common evolutionary pathway.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/011106-0
2008-03-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/3/939.html?itemId=/content/journal/micro/10.1099/mic.0.2007/011106-0&mimeType=html&fmt=ahah

References

  1. Alberti-Segui C., Goeden K. R., Higgins D. E. 2007; Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread. Cell Microbiol 9:179–195
    [Google Scholar]
  2. Arnaud M., Chastanet A., Debarbouille M. 2004; New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol 70:6887–6891
    [Google Scholar]
  3. Autret N., Raynaud C., Dubail I., Berche P., Charbit A. 2003; Identification of the agr locus of Listeria monocytogenes : role in bacterial virulence. Infect Immun 71:4463–4471
    [Google Scholar]
  4. Bannam T., Goldfine H. 1999; Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function. Infect Immun 67:182–186
    [Google Scholar]
  5. Braun L., Nato F., Payrastre B., Mazie J. C., Cossart P. 1999; The 213-amino-acid leucine-rich repeat region of the Listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling. Mol Microbiol 34:10–23
    [Google Scholar]
  6. Braun L., Ghebrehiwet B., Cossart P. 2000; gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes . EMBO J 19:1458–1466
    [Google Scholar]
  7. Cossart P., Vicente M. F., Mengaud J., Baquero F., Perez-Diaz J. C., Berche P. 1989; Listeriolysin O is essential for virulence of Listeria monocytogenes : direct evidence obtained by gene complementation. Infect Immun 57:3629–3636
    [Google Scholar]
  8. Doumith M., Cazalet C., Simoes N., Frangeul L., Jacquet C., Kunst F., Martin P., Cossart P., Glaser P., Buchrieser C. 2004; New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. Infect Immun 72:1072–1083
    [Google Scholar]
  9. Dramsi S., Biswas I., Maguin E., Braun L., Mastroeni P., Cossart P. 1995; Entry of Listeria monocytogenes into hepatocytes requires expression of InlB, a surface protein of the internalin multigene family. Mol Microbiol 16:251–261
    [Google Scholar]
  10. Felicio M. T., Hogg T., Gibbs P., Teixeira P., Wiedmann M. 2007; Recurrent and sporadic Listeria monocytogenes contamination in alheiras represents considerable diversity, including virulence-attenuated isolates. Appl Environ Microbiol 73:3887–3895
    [Google Scholar]
  11. Gaillard J. L., Berche P., Sansonetti P. 1986; Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes . Infect Immun 52:50–55
    [Google Scholar]
  12. Gaillard J. L., Berche P., Frehel C., Gouin E., Cossart P. 1991; Entry of Listeria monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from Gram-positive cocci. Cell 65:1127–1141
    [Google Scholar]
  13. Gracieux P., Roche S. M., Pardon P., Velge P. 2003; Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid′ L. mono media. Int J Food Microbiol 83:133–145
    [Google Scholar]
  14. Handa-Miya S., Kimura B., Takahashi H., Sato M., Ishikawa T., Igarashi K., Fujii T. 2007; Nonsense-mutated inlA and prfA not widely distributed in Listeria monocytogenes isolates from ready-to-eat seafood products in Japan. Int J Food Microbiol 117:312–318
    [Google Scholar]
  15. Jacquet C., Doumith M., Gordon J. I., Martin P. M., Cossart P., Lecuit M. 2004; A molecular marker for evaluating the pathogenic potential of foodborne Listeria monocytogenes . J Infect Dis 189:2094–2100
    [Google Scholar]
  16. Jonquières R., Bierne H., Mengaud J., Cossart P. 1998; The inlA gene of Listeria monocytogenes LO28 harbors a nonsense mutation resulting in release of internalin. Infect Immun 66:3420–3422
    [Google Scholar]
  17. Jonquieres R., Pizarro-Cerda J., Cossart P. 2001; Synergy between the N- and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes . Mol Microbiol 42:955–965
    [Google Scholar]
  18. Kathariou S., Metz P., Hof H., Goebel W. 1987; Tn 916 -induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes . J Bacteriol 169:1291–1297
    [Google Scholar]
  19. Lecuit M., Ohayon H., Braun L., Mengaud J., Cossart P. 1997; Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization. Infect Immun 65:5309–5319
    [Google Scholar]
  20. Lecuit M., Dramsi S., Gottardi C., Fedor-Chaiken M., Gumbiner B., Cossart P. 1999; A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes . EMBO J 18:3956–3963
    [Google Scholar]
  21. Lereclus D., Arantes O. 1992; spbA locus ensures the segregational stability of pTH1030, a novel type of Gram-positive replicon. Mol Microbiol 6:35–46
    [Google Scholar]
  22. Moser J., Gerstel B., Meyer J. E., Chakraborty T., Wehland J., Heinz D. W. 1997; Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes . J Mol Biol 273:269–282
    [Google Scholar]
  23. Nightingale K. K., Windham K., Martin K. E., Yeung M., Wiedmann M. 2005; Select Listeria monocytogenes subtypes commonly found in foods carry distinct nonsense mutations in inlA , leading to expression of truncated and secreted internalin A, and are associated with a reduced invasion phenotype for human intestinal epithelial cells. Appl Environ Microbiol 71:8764–8772
    [Google Scholar]
  24. Norrung B., Andersen J. K. 2000; Variations in virulence between different electrophoretic types of Listeria monocytogenes . Lett Appl Microbiol 30:228–232
    [Google Scholar]
  25. Notermans S. H., Dufrenne J., Leimeister-Wachter M., Domann E., Chakraborty T. 1991; Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl Environ Microbiol 57:2666–2670
    [Google Scholar]
  26. Olier M., Pierre F., Rousseaux S., Lemaitre J. P., Rousset A., Piveteau P., Guzzo J. 2003; Expression of truncated internalin A is involved in impaired internalization of some Listeria monocytogenes isolates carried asymptomatically by humans. Infect Immun 71:1217–1224
    [Google Scholar]
  27. Olier M., Garmyn D., Rousseaux S., Lemaitre J. P., Piveteau P., Guzzo J. 2005; Truncated internalin A and asymptomatic Listeria monocytogenes carriage: in vivo investigation by allelic exchange. Infect Immun 73:644–648
    [Google Scholar]
  28. Park S. F., Stewart G. S. 1990; High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. Gene 94:129–132
    [Google Scholar]
  29. Roche S. M., Velge P., Bottreau E., Durier C., Marquet-van der Mee N., Pardon P. 2001; Assessment of the virulence of Listeria monocytogenes : agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice. Int J Food Microbiol 68:33–44
    [Google Scholar]
  30. Roche S. M., Gracieux P., Albert I., Gouali M., Jacquet C., Martin P. M., Velge P. 2003; Experimental validation of low virulence in field strains of Listeria monocytogenes . Infect Immun 71:3429–3436
    [Google Scholar]
  31. Roche S. M., Gracieux P., Milohanic E., Albert I., Virlogeuz-Payant I., Témoin S., Grépinet O., Kerouanton A., Jacquet C. other authors 2005; Investigation of specific substitutions in virulence genes characterizing phenotypic groups of low-virulence field strains of Listeria monocytogenes . Appl Environ Microbiol 71:6039–6048
    [Google Scholar]
  32. Sabet C., Lecuit M., Cabanes D., Cossart P., Bierne H. 2005; LPXTG protein InlJ, a newly identified internalin involved in Listeria monocytogenes virulence. Infect Immun 73:6912–6922
    [Google Scholar]
  33. Shen Y., Naujokas M., Park M., Ireton K. 2000; InlB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase. Cell 103:501–510
    [Google Scholar]
  34. Tabouret M., De Rycke J., Audurier A., Poutrel B. 1991; Pathogenicity of Listeria monocytogenes isolates in immunocompromised mice in relation to listeriolysin production. J Med Microbiol 34:13–18
    [Google Scholar]
  35. Van Langendonck N., Bottreau E., Bailly S., Tabouret M., Marly J., Pardon P., Velge P. 1998; Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains. J Appl Microbiol 85:337–346
    [Google Scholar]
  36. Vazquez-Boland J. A., Kocks C., Dramsi S., Ohayon H., Geoffroy C., Mengaud J., Cossart P. 1992; Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun 60:219–230
    [Google Scholar]
  37. Velge P., Bottreau E., Van-Langendonck N., Kaeffer B. 1997; Cell proliferation enhances entry of Listeria monocytogenes into intestinal epithelial cells by two proliferation-dependent entry pathways. J Med Microbiol 46:681–692
    [Google Scholar]
  38. Velge P., Herler M., Johansson J., Roche S. M., Témoin S., Fedorov A. A., Gracieux P., Almo S. C., Goebel W., Cossart P. 2007; A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity. Microbiology 153:995–1005
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/011106-0
Loading
/content/journal/micro/10.1099/mic.0.2007/011106-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed