1887

Abstract

We have recently concluded that a 86 kDa immunogenic surface protein, IspC, is a cell wall-anchored peptidoglycan hydrolase (autolysin), capable of degrading the cell wall peptidoglycan of the bacterium itself. To determine if this enzyme has any biological functions and/or plays a role in virulence, we in-frame-deleted the gene from the chromosome. This Δ mutant exhibited complete abrogation of expression of IspC and displayed no defects in growth, colony and microscopic morphologies, or biochemical characteristics. Lack of IspC led to attenuated virulence in mice, evidenced by a significant reduction in bacterial counts in livers and brains and no mortality compared with the wild-type. Furthermore, the data from assays using various eukaryotic cells for adhesion, invasion, actin tail formation, plaque formation and intracellular growth indicated that the mutant was severely attenuated in virulence in a cell culture model in a cell type-dependent manner. The findings that (i) the mutant was impaired for adhesion to certain eukaryotic cells, and (ii) both purified IspC and its C-terminal cell wall-binding domain were capable of binding sheep choroid plexus (SCP) epithelial cells and Vero cells, supported the role of IspC as an adhesin in virulence. The Δ mutant exhibited a marked defect in adhesion to and invasion of SCP cells but not human brain microvascular endothelial cells (HBMEC), suggesting that IspC is necessary for crossing the blood–cerebrospinal fluid barrier. Proteomic and immunological analysis showed a reduced surface expression of some known or putative virulence factors (e.g. ActA, InlC2 and a flagellin homologue, FlaA) due to IspC deficiency. Altogether, this study demonstrates that IspC, expressed as a minor autolysin , is not important for cell division or separation but is essential for full virulence of .

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2008-07-01
2019-10-17
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References

  1. Allignet, J., England, P., Old, I. & El Solh, N. ( 2002; ). Several regions of the repeat domain of the Staphylococcus caprae autolysin, AtlC, are involved in fibronectin binding. FEMS Microbiol Lett 213, 193–197.[CrossRef]
    [Google Scholar]
  2. Alvarez-Dominguez, C., Vazquez-Boland, J. A., Carrasco-Marin, E., Lopez-Mato, P. & Leyva-Cobian, F. ( 1997; ). Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect Immun 65, 78–88.
    [Google Scholar]
  3. Bergmann, B., Raffelsbauer, D., Kuhn, M., Goetz, M., Hom, S. & Goebel, W. ( 2002; ). InlA- but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins. Mol Microbiol 43, 557–570.[CrossRef]
    [Google Scholar]
  4. Berry, A. M. & Paton, J. C. ( 2000; ). Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins. Infect Immun 68, 133–140.[CrossRef]
    [Google Scholar]
  5. Berry, A. M., Lock, R. A., Hansman, D. & Paton, J. C. ( 1989; ). Contribution of autolysin to virulence of Streptococcus pneumoniae. Infect Immun 57, 2324–2330.
    [Google Scholar]
  6. Bierne, H. & Cossart, P. ( 2007; ). Listeria monocytogenes surface proteins: from genome predictions to function. Microbiol Mol Biol Rev 71, 377–397.[CrossRef]
    [Google Scholar]
  7. Braun, L., Dramsi, S., Dehoux, P., Bierne, H., Lindahl, G. & Cossart, P. ( 1997; ). InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association. Mol Microbiol 25, 285–294.[CrossRef]
    [Google Scholar]
  8. Brouwer, M. C., van de Beek, D., Heckenberg, S. G., Spanjaard, L. & de Gans, J. ( 2006; ). Community-acquired Listeria monocytogenes meningitis in adults. Clin Infect Dis 43, 1233–1238.[CrossRef]
    [Google Scholar]
  9. Cabanes, D, Dehoux, P., Dussurget, O., Frangeul, L. & Cossart, P. ( 2002; ). Surface proteins and the pathogenic potential of Listeria monocytogenes. Trends Microbiol 10, 238–245.[CrossRef]
    [Google Scholar]
  10. Cabanes, D., Dussurget, O., Dehoux, P. & Cossart, P. ( 2004; ). Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence. Mol Microbiol 51, 1601–1614.[CrossRef]
    [Google Scholar]
  11. Canvin, J. R., Marvin, A. P., Sivakumaran, M., Paton, J. C., Boulnois, G. J., Andrew, P. W. & Mitchell, T. J. ( 1995; ). The role of pneumolysin and autolysin in the pathology of pneumonia and septicemia in mice infected with a type 2 pneumococcus. J Infect Dis 172, 119–123.[CrossRef]
    [Google Scholar]
  12. Carroll, S. A., Hain, T., Technow, U., Darji, A., Pashalidis, P., Joseph, S. W. & Chakraborty, T. ( 2003; ). Identification and characterization of a peptidoglycan hydrolase, MurA, of Listeria monocytogenes, a muramidase needed for cell separation. J Bacteriol 185, 6801–6808.[CrossRef]
    [Google Scholar]
  13. Dons, L., Eriksson, E., Jin, Y., Rottenberg, M. E., Kristensson, K., Larsen, C. N., Bresciani, J. & Olsen, J. E. ( 2004; ). Role of flagellin and the two-component CheA/CheY system of Listeria monocytogenes in host cell invasion and virulence. Infect Immun 72, 3237–3244.[CrossRef]
    [Google Scholar]
  14. Dramsi, S., Dehoux, P., Lebrun, M., Goossens, P. L. & Cossart, P. ( 1997; ). Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD. Infect Immun 65, 1615–1625.
    [Google Scholar]
  15. Greiffenberg, L., Goebel, W., Kim, K. S., Weiglein, I., Bubert, A., Engelbrecht, F., Stins, M. & Kuhn, M. ( 1998; ). Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells. Infect Immun 66, 5260–5267.
    [Google Scholar]
  16. Heilmann, C., Hussain, M., Peters, G. & Gotz, F. ( 1997; ). Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 24, 1013–1024.[CrossRef]
    [Google Scholar]
  17. Hell, W., Meyer, H. G. & Gatermann, S. G. ( 1998; ). Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties. Mol Microbiol 29, 871–881.[CrossRef]
    [Google Scholar]
  18. Jedrzejas, M. J. ( 2001; ). Pneumococcal virulence factors: structure and function. Microbiol Mol Biol Rev 65, 187–207.[CrossRef]
    [Google Scholar]
  19. Kazmierczak, M. J., Mithoe, S. C., Boor, K. J. & Wiedmann, M. ( 2003; ). Listeria monocytogenes σ B regulates stress response and virulence functions. J Bacteriol 185, 5722–5734.[CrossRef]
    [Google Scholar]
  20. Lenz, L. L., Mohammadi, S., Geissler, A. & Portnoy, D. A. ( 2003; ). SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc Natl Acad Sci U S A 100, 12432–12437.[CrossRef]
    [Google Scholar]
  21. Lock, R. A., Hansman, D. & Paton, J. C. ( 1992; ). Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae. Microb Pathog 12, 137–143.[CrossRef]
    [Google Scholar]
  22. MacFaddin, J. F. ( 2000; ). Biochemical Tests for Identification of Medical Bacteria, 3rd edn. New York: Lippincott Williams & Wilkins.
  23. Machata, S., Hain, T., Rohde, M. & Chakraborty, T. ( 2005; ). Simultaneous deficiency of both MurA and p60 proteins generates a rough phenotype in Listeria monocytogenes. J Bacteriol 187, 8385–8394.[CrossRef]
    [Google Scholar]
  24. Mani, N., Baddour, L. M., Offutt, D. Q., Vijaranakul, U., Nadakavukaren, M. J. & Jayaswal, R. K. ( 1994; ). Autolysis-defective mutant of Staphylococcus aureus: pathological considerations, genetic mapping, and electron microscopic studies. Infect Immun 62, 1406–1409.
    [Google Scholar]
  25. Milohanic, E., Jonquieres, R., Cossart, P., Berche, P. & Gaillard, J. L. ( 2001; ). The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor. Mol Microbiol 39, 1212–1224.[CrossRef]
    [Google Scholar]
  26. O'Neil, H. S. & Marquis, H. ( 2006; ). Listeria monocytogenes flagella are used for motility, not as adhesins, to increase host cell invasion. Infect Immun 74, 6675–6681.[CrossRef]
    [Google Scholar]
  27. Park, S. F. & Stewart, G. S. ( 1990; ). High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. Gene 94, 129–132.[CrossRef]
    [Google Scholar]
  28. Pilgrim, S., Kolb-Maurer, A., Gentschev, I., Goebel, W. & Kuhn, M. ( 2003; ). Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility. Infect Immun 71, 3473–3484.[CrossRef]
    [Google Scholar]
  29. Popowska, M. ( 2004; ). Analysis of the peptidoglycan hydrolases of Listeria monocytogenes: multiple enzymes with multiple functions. Pol J Microbiol 53 (suppl.), 29–34.
    [Google Scholar]
  30. Popowska, M. & Markiewicz, Z. ( 2004; ). Murein-hydrolyzing activity of flagellin FlaA of Listeria monocytogenes. Pol J Microbiol 53, 237–241.[CrossRef]
    [Google Scholar]
  31. Prats, N., Briones, V., Blanco, M. M., Altimira, J., Ramos, J. A., Dominguez, L. & Marco, A. ( 1992; ). Choroiditis and meningitis in experimental murine infection with Listeria monocytogenes. Eur J Clin Microbiol Infect Dis 11, 744–747.[CrossRef]
    [Google Scholar]
  32. Rowan, N. J., Candlish, A. A., Bubert, A., Anderson, J. G., Kramer, K. & McLauchlin, J. ( 2000; ). Virulent rough filaments of Listeria monocytogenes from clinical and food samples secreting wild-type levels of cell-free p60 protein. J Clin Microbiol 38, 2643–2648.
    [Google Scholar]
  33. Rupp, M. E., Ulphani, J. S., Fey, P. D., Bartscht, K. & Mack, D. ( 1999; ). Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infect Immun 67, 2627–2632.
    [Google Scholar]
  34. Rupp, M. E., Fey, P. D., Heilmann, C. & Gotz, F. ( 2001; ). Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model. J Infect Dis 183, 1038–1042.[CrossRef]
    [Google Scholar]
  35. Schaferkordt, S., Domann, E. & Chakraborty, T. ( 1998; ). Molecular approaches for the study of Listeria. In Bacterial Pathogenesis (Methods in Microbiology no. 27) pp. 421–431. Edited by P. Williams, J. Ketley & G. Salmond. San Diego, CA: Academic Press.
  36. Schluter, D., Chahoud, S., Lassmann, H., Schumann, A., Hof, H. & Deckert-Schluter, M. ( 1996; ). Intracerebral targets and immunomodulation of murine Listeria monocytogenes meningoencephalitis. J Neuropathol Exp Neurol 55, 14–24.[CrossRef]
    [Google Scholar]
  37. Shockman, G. D. & Holtje, J.-V. ( 1994; ). Microbial peptidoglycan (murein) hydrolases. In Bacterial Cell Wall, pp. 131–166. Edited by J.-M. Ghuysen & R. Hakenbeck. Amsterdam: Elsevier.
  38. Smith, T. J., Blackman, S. A. & Foster, S. J. ( 2000; ). Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146, 249–262.
    [Google Scholar]
  39. Takahashi, J., Komatsuzawa, H., Yamada, S., Nishida, T., Labischinski, H., Fujiwara, T., Ohara, M., Yamagishi, J. & Sugai, M. ( 2002; ). Molecular characterization of an atl null mutant of Staphylococcus aureus. Microbiol Immunol 46, 601–612.[CrossRef]
    [Google Scholar]
  40. Tuomanen, E. ( 1996; ). Entry of pathogens into the central nervous system. FEMS Microbiol Rev 18, 289–299.[CrossRef]
    [Google Scholar]
  41. Tuomanen, E. I. ( 2000; ). Pathogenesis of pneumococcal inflammation: otitis media. Vaccine 19 (suppl. 1), S38–S40.[CrossRef]
    [Google Scholar]
  42. Vandegraaff, R., Borland, N. A. & Browning, J. W. ( 1981; ). An outbreak of listerial meningo-encephalitis in sheep. Aust Vet J 57, 94–96.[CrossRef]
    [Google Scholar]
  43. Vasilescu, J., Smith, J. C., Ethier, M. & Figeys, D. ( 2005; ). Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. J Proteome Res 4, 2192–2200.[CrossRef]
    [Google Scholar]
  44. Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J. & Kreft, J. ( 2001; ). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584–640.[CrossRef]
    [Google Scholar]
  45. Wang, L. & Lin, M. ( 2007; ). Identification of IspC, an 86-kilodalton protein target of humoral immune response to infection with Listeria monocytogenes serotype 4b, as a novel surface autolysin. J Bacteriol 189, 2046–2054.[CrossRef]
    [Google Scholar]
  46. Wang, L., Walrond, L., Cyr, T. D. & Lin, M. ( 2007; ). A novel surface autolysin of Listeria monocytogenes serotype 4b, IspC, contains a 23-residue N-terminal signal peptide being processed in E. coli. Biochem Biophys Res Commun 354, 403–408.[CrossRef]
    [Google Scholar]
  47. Yu, W. L., Dan, H. & Lin, M. ( 2007; ). Novel protein targets of humoral immune response to Listeria monocytogenes infection in rabbits. J Med Microbiol 56, 888–895.[CrossRef]
    [Google Scholar]
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