Tissue Culture Cell Assays Used to Analyze Listeria monocytogenes

Hélène Marquis1

1 Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9B.4
DOI:  10.1002/9780471729259.mc09b04s01
Online Posting Date:  June, 2006
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Abstract

This unit describes tissue culture cell assays for analysis of the ability of Listeria monocytogenes to cause intracellular infection. It includes methods for evaluating the organism's ability to invade its host, to escape the primary vacuole formed upon invasion of host cells, to multiply within the cytosol of its host, and to spread from cell to cell without exiting the intracellular milieu. Each step can be evaluated quantitatively and qualitatively.

Keywords: invasion; escape from vacuole; intracellular growth; cell‐to‐cell spread; Listeria

     
 
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Table of Contents

  • Basic Protocol 1: Host Cell Invasion
  • Basic Protocol 2: Escape from Vacuoles
  • Basic Protocol 3: Kinetics of Intracellular Growth
  • Basic Protocol 4: Cell‐to‐Cell Spread
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Host Cell Invasion

  Materials
  • 95% ethanol
  • 100 µg/ml type I rat tail collagen (see recipe)
  • CaCl 2 and MgCl 2‐free Dulbecco's PBS (CMF‐DPBS; GIBCO, also see appendix 2A), sterile
  • Caco‐2 cells (ATCC #HTB‐37), trypsinized
  • Caco‐2 medium (see recipe), 4° and 37°C
  • BHI medium (see recipe)
  • H 2O, sterile
  • Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researchers working with L. monocytogenes, or clinical isolates)
  • LB plates ( appendix 4A)
  • 10 mg/ml gentamicin (see recipe)
  • Diff‐Quick stain set (Dade Behring)
  • Permount (Fisher)
  • 12‐mm round, precleaned glass coverslips in 70% ethanol
  • 35‐mm tissue culture–treated dishes (BD Falcon)
  • Pasteur pipets, sterile
  • 15‐ and 50‐ml polypropylene conical tubes, sterile
  • 37°C, 5% CO 2 humidified incubator
  • 37°C water bath
  • 30° and 37°C incubators
  • 1.5‐ml microcentrifuge tubes, sterile
  • 15‐ml polystyrene conical tubes, sterile
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 4A)

Basic Protocol 2: Escape from Vacuoles

  Materials
  • 95% ethanol
  • J774 cells (ATCC #TIB‐67)
  • J774 medium with antibiotics (see recipe for J774 and L2 medium), 37°C
  • CaCl 2‐ and MgCl 2‐free Dulbecco's PBS (DMF‐DPBS, GIBCO; also see appendix 2A), sterile, 4°C and room temperature
  • J774 medium without antibiotics, 4° and 37°C
  • BHI medium (see recipe)
  • Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researchers working with L. monocytogenes, or clinical isolates)
  • 10 mg/ml gentamicin (see recipe)
  • Formalin (see recipe)
  • TBS‐TX (see recipe)
  • Antibody buffer (see recipe)
  • Rabbit anti‐Listeria antibody (Oxoid)
  • Secondary antibodies (e.g., goat anti‐rabbit IgG conjugated to an Alexa Fluor 350 and 568, Invitrogen‐Molecular Probes)
  • Alexa Fluor 488 phalloidin (Invitrogen‐Molecular Probes)
  • TBS (see recipe)
  • Prolong Antifade (Invitrogen‐Molecular Probes)
  • 18‐mm square pre‐cleaned glass coverslips in 70% ethanol
  • 35‐mm tissue culture–treated dishes (BD Falcon)
  • 75‐cm2 non‐tissue culture–treated flasks
  • Pasteur pipets, sterile
  • 15‐ and 50‐ml sterile polypropylene conical tubes
  • Refrigerated tabletop swinging bucket centrifuge, 4°C
  • 37°C, 5% CO 2 humidified incubator
  • 30°C incubator
  • 1.5‐ml microcentrifuge tubes, sterile
  • 37°C water bath
  • 150‐mm petri dishes
  • Fluorescence microscope (filter sets for blue, green, and red fluorophores)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 4A)

Basic Protocol 3: Kinetics of Intracellular Growth

  Materials
  • 95% ethanol
  • J774 cells (ATCC #TIB‐67)
  • J774 medium with antibiotics (see recipe for J774 and L2 medium), 37°C
  • CaCl 2‐ and MgCl 2‐free Dulbecco's PBS (CMF‐DPBS, GIBCO; also see appendix 2A), sterile, 4°C and room temperature
  • J774 medium without antibiotics, 4° and 37°C
  • BHI medium (see recipe)
  • Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researcher working with L. monocytogenes, or clinical isolates)
  • 10 mg/ml gentamicin (see recipe)
  • LB plates ( appendix 4A)
  • Diff‐Quick stain set (Dade Behring)
  • Permount (Fisher)
  • 12‐mm round pre‐cleaned glass coverslips in 70% ethanol
  • 60‐mm petri dishes (Nunc)
  • 75‐cm2 nontissue culture–treated flasks
  • Pasteur pipets, sterile
  • 15‐ and 50‐ml polypropylene conical tubes, sterile
  • Refrigerated tabletop swinging‐bucket centrifuge, 4°C
  • 37°C, 5% CO 2 incubator
  • 30° and 37°C incubators
  • 1.5‐ml microcentrifuge tubes, sterile
  • 37°C water bath
  • 15‐ml sterile polystyrene conical tubes
  • Plotting software (e.g., Prism, Cricket Graph, or Excel)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 4A)

Basic Protocol 4: Cell‐to‐Cell Spread

  Materials
  • L2 mouse fibroblast cell line (individual researchers working with Corona viruses or with L. monocytogenes), trypsinized
  • L2 medium (see recipe for J774 and L2 medium) without antibiotics, 4°C
  • BHI medium (see recipe)
  • Bacterial strain(s) (e.g., L. monocytogenes; ATCC, individual researchers working with L. monocytogenes or clinical isolates, sterile)
  • Dulbecco's PBS CaCl 2‐ and MgCl 2‐free (CMF‐DPBS, GIBCO; see appendix 2A)
  • L. monocytogenes plaquing medium (see recipe), 37°C
  • 10 mg/ml gentamicin (see recipe)
  • 1.4% (w/v) agarose: autoclave and allow to harden
  • 1 N HCl
  • 0.5% neutral red solution, pH 5.2 (Sigma)
  • 15‐ and 50‐ml conical polypropylene tubes, sterile
  • 6‐well tissue culture–treated plates or 35‐mm tissue culture–treated dishes
  • 37°C, 5% CO 2 humidified incubator
  • 30°C incubator
  • 1.5‐ml microcentrifuge tubes, sterile
  • 37°C water bath
  • 50° to 55°C water bath
  • Pasteur pipets, sterile
  • Scanner
  • Plaque measuring software: Adobe Photoshop or equivalent
  • Additional reagents and solutions for counting cells in a hemacytometer ( appendix 4A)
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Figures

Videos

Literature Cited

Literature Cited
   Bakardjiev, A.I., Stacy, B.A., Fisher, S.J., and Portnoy, D.A. 2004. Listeriosis in the pregnant guinea pig: A model of vertical transmission. Infect. Immun. 72:489‐497.
   Bishop, D.K. and Hinrichs, D.J. 1987. Adoptive transfer of immunity to Listeria monocytogenes: The influence of in vitro stimulation on lymphocyte subset requirements. J. Immunol. 139:2005‐2009.
   CDC. 1999. Multistate outbreak of listeriosis—United States, 1998‐1999. JAMA 281:317‐318.
   Dales, S. 1962. An electron microscope study of the early association between two mammalian viruses and their hosts. J. Cell. Biol. 13:303‐322.
   Dussurget, O., Pizarro‐Cerda, J., and Cossart, P. 2004. Molecular determinants of Listeria monocytogenes virulence. Annu. Rev. Microbiol. 58:587‐610.
   Fenlon, D.R. 1999. Listeria monocytogenes in the natural environment. In Listeria, listeriosis, and food safety, 2nd ed. (E. T. Ryser and E. H. Marth, eds.) pp. 21‐37. Marcel Dekker, New York.
   Gaillard, J.‐L., Berche, P., Frehel, C., Gouin, E., and 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.
   Gaillard, J.‐L., Berche, P., Mounier, J., Richard, S., and 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.
   Gaillard, J.‐L., Jaubert, F., and Berche, P. 1996. The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. J. Exp. Med. 183:359‐369.
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   Lenz, L.L. and Portnoy, D.A. 2002. Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype. Mol. Microbiol. 45:1043‐1056.
   Marquis, H., Doshi, V., and Portnoy, D.A. 1995. The broad‐range phospholipase C and a metalloprotease mediate listeriolysin O‐independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect. Immun. 63:4531‐4534.
   Mounier, J., Ryter, A., Coquis‐Rondon, M., and Sansonetti, P.J. 1990. Intracellular and cell‐to‐cell spread of Listeria monocytogenes involves interaction with F‐actin in the enterocytelike cell line Caco‐2. Infect. Immun. 58:1048‐1058.
   Portnoy, D.A., Chakraborty, T., Goebel, W., and Cossart, P. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263‐1267.
   Portnoy, D.A., Jacks, P.S., and Hinrichs, D.J. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167:1459‐1471.
   Sun, A.N., Camilli, A., and Portnoy, D.A. 1990. Isolation of Listeria monocytogenes small‐plaque mutants defective for intracellular growth and cell‐to‐cell spread. Infect. Immun. 58:3770‐3778.
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   Vazquez‐Boland, J.A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez‐Bernal, G., Goebel, W., Gonzalez‐Zorn, B., Wehland, J., and Kreft, J. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584‐640.
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