Animal Model for Infection with Listeria monocytogenes

Dirk H. Busch1, Sujata Vijh1, Eric G. Pamer1

1 Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.9
DOI:  10.1002/0471142735.im1909s36
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for infecting mice with L. monocytogenes. Optimal media for growth and methods to maintain bacterial virulence by passage through mice are included. Methods for determining the severity of splenic and hepatic infection are detailed, with strategies for distinguishing innate from specific immune responses following L. monocytogenes infection. This infection induces MHC class I‐restricted CD8+ cytolytic T lymphocytes that clear infection and provide long‐term immunity. This unit describes methods that can be used for in vitro expansion of L. monocytogenes‐specific T cells.

     
 
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Table of Contents

  • Basic Protocol 1: Infection of Mice with L. monocytogenes
  • Support Protocol 1: Evaluation of Specific and Innate Immune Responses to L. Monocytogenes in Mice
  • Support Protocol 2: Generation of Listeria‐Specific T Cells
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Infection of Mice with L. monocytogenes

  Materials
  • L. monocytogenes stocks (units 14.6 & 16.1)
  • Brain heart infusion (BHI) broth (Difco), sterile
  • PBS ( appendix 2A)
  • Mice (Table 19.9.2)
    Table 9.9.2   Materials   Susceptibility of Different Mouse Strains to Infection with Listeria monocytogenes c   Susceptibility of Different Mouse Strains to Infection with Listeria monocytogenes

    Susceptible Intermediate Resistant
    BALB/c (C57BL/6 × BALB/c) F 1 C57BL/6
    C3H/HeJ B10.D2
    129/J B10.A
    DBA2 B6.PL
    CBA/H SJL/WEHI

     cData from Cheers and McKenzie ( ).
  • Spectrophotometer
  • Infrared heating lamp
  • Mouse restraining device
  • 1‐ml syringe
  • 27‐G needle
  • Additional reagents and equipment for preparing L. monocytogenes stocks (units 14.6 & 16.1) and performing i.v. injections (unit 1.6)

Support Protocol 1: Evaluation of Specific and Innate Immune Responses to L. Monocytogenes in Mice

  • Naive (nonimmunized) mice (optional)
  • 0.05% (v/v) Triton X‐100
  • Brain heart infusion (BHI) agar (Difco) plates, sterile
  • Stainless steel wire mesh screens
  • 50‐mm sterile Petri dishes
  • Dissecting forceps and scissors, sterile
  • 5‐ml plastic syringes, sterile
  • 12 × 75–mm polystyrene tubes, sterile
  • Glass spreader, sterile
  • Additional reagents and equipment for euthanasia (unit 1.8)

Support Protocol 2: Generation of Listeria‐Specific T Cells

  • J774 macrophage‐like cells (ATCC #TIB 67)
  • Complete RPMI‐10 ( appendix 2A) without antibiotics
  • Complete RPMI‐10 containing 5 µg/ml gentamicin (in place of other antibiotics)
  • L. monocytogenes‐infected mice (see protocol 1)
  • Spleen filler cells
  • Rat concanavalin A (Con A)–activated supernatant (unit 3.13)
  • 1 M α‐methylmannoside
  • Naive (uninfected) syngeneic BALB/c mice
  • 1 mM synthetic peptide stock solution (optional; see Table 19.9.3)
  • 10‐cm tissue culture plates (e.g., Falcon)
  • 25‐cm2 tissue culture flasks (e.g., Falcon)
  • γ irradiator
  • 0.22‐µm syringe filter (optional)
  • Additional reagents and equipment for counting cells by trypan blue exclusion ( appendix 3B) and with a hemacytometer ( appendix 3A), for preparing splenocytes (unit 3.1), and for subcloning by limiting dilution (unit 3.13)
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Figures

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Literature Cited

Literature Cited
   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.
   Busch, D.H. and Pamer, E.G. 1998. MHC class I/peptide stability: Implications for immunodominance, in vitro proliferation and diversity of responding CTL. J. Immunol. 160:4441‐4448.
   Busch, D.H., Pilip, I.M., Vijh, S., and Pamer, E.G. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8:353‐362.
   Cheers, C. and McKenzie, I. 1978. Resistance and susceptibility of mice to bacterial infection: Genetics of Listeriosis. Infect. Immun. 19:755‐762.
   Cossart, P. and Mengaud, J. 1989. Listeria monocytogenes: A model system for the molecular study of intracellular parasitism. Mol. Biol. Med. 6:463‐474.
   Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister‐Wachter, M., and Chakraborty, T. 1992. 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. 11:1981‐1990.
   Gulden, P.H., Fischer, P., Sherman, N.E., Wang, W., Engelhard, V.H., Shabanowitz, J., Hunt, D.H., and Pamer, E.G. 1996. A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2‐M3 MHC class Ib molecule. Immunity 5:73‐79.
   Harty, J.T. and Bevan, M.J. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN gamma. Immunity 3:109‐118.
   Kaufmann, S.H.E. 1995. Immunity to intracellular microbial pathogens. Immunol. Today 16:338‐342.
   Kaufmann, S.H.E., Hug, E., and DeLibero, G. 1986. Listeria monocytogenes reactive T lymphocyte clones with cytolytic activity against infected target cells. J. Exp. Med. 164:363‐368.
   Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H., and Cossart, P. 1992. L. monocytogenes‐induced actin assembly requires the actA gene product, a surface protein. Cell 68:521‐531.
   Ladel, C.H., Flesch, I.E.F., Arnoldi, J., and Kaufmann, S.H.E. 1994. Studies with MHC‐deficient knock‐out mice reveal impact of both MHC I‐ and MHC II‐dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153:3116‐3122.
   Lenz, L.L., Dere, B., and Bevan, M.J. 1996. Identification of an H2‐M3‐restricted Listeria epitope: Implications for antigen presentation by M3. Immunity 5:63‐72.
   Mackaness, G.B. 1962. Cellular resistance to infection. J. Exp. Med. 116:381‐406.
   Pamer, E.G. 1997. Immune response to Listeria monocytogenes. In Host Response to Intracellular Pathogens (S.H.E. Kaufmann, ed.) pp.131‐142. R.G. Landes, Austin, Tex.
   Portnoy, D.A., Chakraborty, T., Goebel, W., and Cossart, P. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263‐1267.
   Princiotta, M.F., Lenz, L.L., Bevan, M.J., and Staerz, U.D. 1998. H2‐M3 restricted presentation of a Listeria‐derived leader peptide. J. Exp. Med. 187:1711‐1719.
   Unanue, E. 1997. Inter‐relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance. Curr. Opin. Immunol. 9:35‐43.
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