Skip to main content
Infection and Immunity logoLink to Infection and Immunity
. 1990 Apr;58(4):1048–1058. doi: 10.1128/iai.58.4.1048-1058.1990

Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2.

J Mounier 1, A Ryter 1, M Coquis-Rondon 1, P J Sansonetti 1
PMCID: PMC258581  PMID: 2108086

Abstract

Listeria monocytogenes penetrates and multiplies within professional phagocytes and other cells such as the Caco-2 human enterocytelike cell line. Listeriolysin O, a membrane-damaging cytotoxin accounts for intracellular multiplication through lysis of the membrane-bound phagocytic vacuole. This work demonstrates that once released within the cytosol, L. monocytogenes acquires the capacity to spread intracellularly and infect adjacent cells by interacting with host cell microfilaments. Such evidence was obtained by using drugs which disrupt the cell cytoskeleton. Nocodazole, which blocks polymerization of microtubules, did not affect intracellular spread, whereas cytochalasin D, which blocks polymerization of G-actin, inhibited the intracellular motility of the bacteria. By using fluorescence staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), transmission electron microscopy, and immunogold labeling, direct evidence was obtained that intracellular bacteria were enveloped with a thick layer of F-actin. Within 2 h after entry, it was demonstrated by confocal microscopy that bacteria were following highly organized routes corresponding to stress fibers. Four hours after entry, some bacteria presented random movements which could be seen by the presence of a large trail of F-actin. Such movements also caused protrusions which deeply penetrated adjacent cells and resulted in the formation of vacuoles limited by a double membrane. After subsequent lysis of these membranes, bacteria released within the cytoplasm were able to multiply and invade new cells. In contrast, an hly::Tn1545 mutant of the wild-type microorganism demonstrated almost no intracellular spread. Only a few bacteria displaying delayed lysis of the phagocytic vacuole behaved like the wild-type strain. Hemolysin-mediated lysis of the phagocytic vacuole and subsequent interaction with host cell microfilaments may represent a major virulence factor allowing tissue colonization during listeriosis.

Full text

PDF
1048

Images in this article

Selected References

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

  1. Barak L. S., Yocum R. R., Nothnagel E. A., Webb W. W. Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. Proc Natl Acad Sci U S A. 1980 Feb;77(2):980–984. doi: 10.1073/pnas.77.2.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baudry B., Maurelli A. T., Clerc P., Sadoff J. C., Sansonetti P. J. Localization of plasmid loci necessary for the entry of Shigella flexneri into HeLa cells, and characterization of one locus encoding four immunogenic polypeptides. J Gen Microbiol. 1987 Dec;133(12):3403–3413. doi: 10.1099/00221287-133-12-3403. [DOI] [PubMed] [Google Scholar]
  3. Bernardini M. L., Mounier J., d'Hauteville H., Coquis-Rondon M., Sansonetti P. J. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A. 1989 May;86(10):3867–3871. doi: 10.1073/pnas.86.10.3867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chailley B., Bork K., Gounon P., Sandoz D. Immunological detection of actin in isolated cilia from quail oviduct. Biol Cell. 1986;58(1):43–52. doi: 10.1111/j.1768-322x.1986.tb00487.x. [DOI] [PubMed] [Google Scholar]
  5. Clerc P., Baudry B., Sansonetti P. J. Plasmid-mediated contact haemolytic activity in Shigella species: correlation with penetration into HeLa cells. Ann Inst Pasteur Microbiol. 1986 May-Jun;137A(3):267–278. doi: 10.1016/s0769-2609(86)80033-3. [DOI] [PubMed] [Google Scholar]
  6. Clerc P., Sansonetti P. J. Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect Immun. 1987 Nov;55(11):2681–2688. doi: 10.1128/iai.55.11.2681-2688.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ewing E. P., Jr, Takeuchi A., Shirai A., Osterman J. V. Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study. Infect Immun. 1978 Mar;19(3):1068–1075. doi: 10.1128/iai.19.3.1068-1075.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Finlay B. B., Gumbiner B., Falkow S. Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer. J Cell Biol. 1988 Jul;107(1):221–230. doi: 10.1083/jcb.107.1.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fleming D. W., Cochi S. L., MacDonald K. L., Brondum J., Hayes P. S., Plikaytis B. D., Holmes M. B., Audurier A., Broome C. V., Reingold A. L. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N Engl J Med. 1985 Feb 14;312(7):404–407. doi: 10.1056/NEJM198502143120704. [DOI] [PubMed] [Google Scholar]
  10. Gaillard J. L., Berche P., Mounier J., Richard S., Sansonetti P. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun. 1987 Nov;55(11):2822–2829. doi: 10.1128/iai.55.11.2822-2829.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaillard J. L., Berche P., Sansonetti P. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect Immun. 1986 Apr;52(1):50–55. doi: 10.1128/iai.52.1.50-55.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geoffroy C., Gaillard J. L., Alouf J. E., Berche P. Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect Immun. 1987 Jul;55(7):1641–1646. doi: 10.1128/iai.55.7.1641-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gray M. L., Killinger A. H. Listeria monocytogenes and listeric infections. Bacteriol Rev. 1966 Jun;30(2):309–382. doi: 10.1128/br.30.2.309-382.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Havell E. A. Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes. Infect Immun. 1986 Dec;54(3):787–792. doi: 10.1128/iai.54.3.787-792.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Isberg R. R., Falkow S. A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. Nature. 1985 Sep 19;317(6034):262–264. doi: 10.1038/317262a0. [DOI] [PubMed] [Google Scholar]
  16. Kathariou S., Metz P., Hof H., Goebel W. Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J Bacteriol. 1987 Mar;169(3):1291–1297. doi: 10.1128/jb.169.3.1291-1297.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kuhn M., Goebel W. Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infect Immun. 1989 Jan;57(1):55–61. doi: 10.1128/iai.57.1.55-61.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MACKANESS G. B. Cellular resistance to infection. J Exp Med. 1962 Sep 1;116:381–406. doi: 10.1084/jem.116.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Makino S., Sasakawa C., Kamata K., Kurata T., Yoshikawa M. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell. 1986 Aug 15;46(4):551–555. doi: 10.1016/0092-8674(86)90880-9. [DOI] [PubMed] [Google Scholar]
  20. Miller V. L., Finlay B. B., Falkow S. Factors essential for the penetration of mammalian cells by Yersinia. Curr Top Microbiol Immunol. 1988;138:15–39. [PubMed] [Google Scholar]
  21. Moulder J. W. Comparative biology of intracellular parasitism. Microbiol Rev. 1985 Sep;49(3):298–337. doi: 10.1128/mr.49.3.298-337.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. NJOKU-OBI A. N., JENKINS E. M., NJOKU-OBI J. C., ADAMS J., COVINGTON V. PRODUCTION AND NATURE OF LISTERIA MONOCYTOGENES HEMOLYSINS. J Bacteriol. 1963 Jul;86:1–8. doi: 10.1128/jb.86.1.1-8.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Portnoy D. A., Jacks P. S., Hinrichs D. J. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med. 1988 Apr 1;167(4):1459–1471. doi: 10.1084/jem.167.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rousset M. The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation. Biochimie. 1986 Sep;68(9):1035–1040. doi: 10.1016/s0300-9084(86)80177-8. [DOI] [PubMed] [Google Scholar]
  25. Sansonetti P. J., Arondel J. Construction and evaluation of a double mutant of Shigella flexneri as a candidate for oral vaccination against shigellosis. Vaccine. 1989 Oct;7(5):443–450. doi: 10.1016/0264-410x(89)90160-6. [DOI] [PubMed] [Google Scholar]
  26. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun. 1986 Feb;51(2):461–469. doi: 10.1128/iai.51.2.461-469.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  28. Tilney L. G., Portnoy D. A. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J Cell Biol. 1989 Oct;109(4 Pt 1):1597–1608. doi: 10.1083/jcb.109.4.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vale R. D. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol. 1987;3:347–378. doi: 10.1146/annurev.cb.03.110187.002023. [DOI] [PubMed] [Google Scholar]
  30. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]
  31. Wassef J. S., Keren D. F., Mailloux J. L. Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis. Infect Immun. 1989 Mar;57(3):858–863. doi: 10.1128/iai.57.3.858-863.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Whitehouse R. L., Benichou J. C., Couture-Tosi E., Schenkman S., Ryter A. Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E. coli embedded in Lowicryl. Biol Cell. 1984;51(3):389–394. doi: 10.1111/j.1768-322x.1984.tb00314.x. [DOI] [PubMed] [Google Scholar]
  33. Winkler H. H., Turco J. Rickettsia prowazekii and the host cell: entry, growth and control of the parasite. Curr Top Microbiol Immunol. 1988;138:81–107. [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES