Tissue Culture Assays Used to Analyze Invasion by Staphylococcus aureus

Ambrose L. Cheung1, Kenneth W. Bayles2

1 Dartmouth Medical School, Hanover, New Hampshire, 2 University of Nebraska Medical Center, Omaha, Nebraska
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9C.4
DOI:  10.1002/9780471729259.mc09c04s4
Online Posting Date:  February, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Many successful pathogens have developed the ability to adhere to and invade animal tissues. Recent experimental evidence suggests that S. aureus, generally perceived as an extracellular pathogen, can also invade and, in some cases, multiply within host cells. As a proxy to infections in animal hosts, the study of S. aureus interactions with tissue culture cells has become an important research tool in many aspects of bacterial pathogenesis. In this unit, we describe two cell culture models, including bovine mammary epithelial cells and human umbilical vein endothelial cells, that investigators have used to study the interactions of S. aureus with host cells.

Keywords: Staphylococcus aureus; cell culture; adhesins; invasion; bovine mammary epithelial cells; human umbilical vein endothelial cells

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Standard S. aureus Invasion of MAC‐T Cells
  • Basic Protocol 2: Assay for Adherence of S. aureus to Cultured Human Umbilical Vein Endothelial Cells (HUVECs)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Standard S. aureus Invasion of MAC‐T Cells

  Materials
  • MAC‐T cells (contact Kenneth W. Bayles, )
  • MAC‐T growth medium (see recipe)
  • 0.25% trypsin in HBSS (Sigma)
  • Invasion medium: MAC‐T growth medium (see recipe) without antibiotics or FBS
  • S. aureus (strain RN6390; Network on Antimicrobial Resistance in Staphylococcus aureus; http://www.narsa.net)
  • Tryptic soy broth (TSB; see recipe) as bacterial growth medium
  • Hanks' balanced salt solution (HBSS; appendix 2A), filter sterilized
  • Gentamicin sulfate (Invitrogen)
  • 0.025% (v/v) Triton X‐100 (U.S. Biochemical) in sterile distilled water
  • Tryptic soy agar plates (see recipe)
  • 24‐well tissue culture plates (Costar)
  • Shaking bacteriological incubator
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 4A)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Assay for Adherence of S. aureus to Cultured Human Umbilical Vein Endothelial Cells (HUVECs)

  Materials
  • S. aureus (strain RN6390; Network on Antimicrobial Resistance in Staphylococcus aureus; http://www.narsa.net)
  • Bacterial growth medium: e.g., tryptic soy broth (TSB; see recipe) or brain‐heart infusion medium (e.g., Difco)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • M199 medium (Bio‐Whittaker)
  • Tryptic soy agar for growing S. aureus (see recipe)
  • Human umbilical vein epithelial cells (HUVEC) grown to confluence in gelatin‐coated 96‐well plates ( appendix 4B)
  • 0.05% (w/v) collagenase/0.01% (w/v) EDTA
  • 0.25% trypsin/0.1% EDTA (Cellgro) in M199 medium (Bio‐Whittaker)
  • 2% (v/v) Triton X‐100 in H 2O
  • 10 µg/ml lysostaphin (Ambi Biologicals)
  • 18‐mm glass culture tube
  • Humidified 37°C, 5% CO 2 incubator
  • Microtip sonicator (e.g., Branson)
  • Additional reagents and equipment for culture of HUVEC ( appendix 4B) and trypan blue exclusion test of cell viability (Strober, )
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: Unless otherwise stated, all reagents and incubations are at 37°C.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bayles, K.W., Wesson, C.A., Liou, L.E., Fox, L.K., Bohach, G.A., and Trumble, W.R. 1998. Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis in epithelial cells. Infect. Immun. 66:336‐342.
   Cheung, A.L., Krishnan, M., Jaffe, E.A., and Fischetti, V.A. 1991. Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells. J. Clin. Invest. 87:2236‐2245.
   Cheung, A.L., Bayer, A.S., Zhang, G., Gresham, H., and Xiong, Y.‐Q. 2004. Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus. FEMS Microbiol. Lett. 1649:1‐9.
   Facchinetti, A., Tessarollo, L., Mazzocchi, M., Kingston, R., Collavo, D., and Biasi, G. 1991. An improved method for the detection of DNA fragmentation. J. Immunol. Methods 136:1251‐1256.
   Fox, L.K., Bayles, K.W., and Bohach, G.A. 2000. Staphylococcus aureus mastitis. In Staphylococcal Infection and Immunity (A. Honeyman, H. Friedman, and M. Bendinelli, eds.) pp. 271‐294. Kluwer Academic/Plenum Publishers, New York.
   Gavrieli, Y., Sherman, Y., and Ben‐Sasson, S.A. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493‐501.
   Hyunh, H.T., Robitaille, G., and Turner, J.D. 1991. Establishment of bovine mammary epithelial cells (MAC‐T): An in vivo model for bovine lactation. Exp. Cell Res. 197:191‐199.
   Jaffe, E.A. 1980. Culture of human endothelial cells. Transplant. Proc. 12:49‐53.
   Jaffe, E.A., Nachman, R.L., Becker, C.G., and Minick, C.R. 1973. Culture of human endothelial cells derived from umbilical veins: Identification by morphologic and immunologic criteria. J. Clin. Invest. 52:2745‐2756.
   Kahl, B., Goulian, M., Van Wamel, W., Herrmann, M., Simon, S., Kaplan, G. et al. 2000. Staphylococcus aureus RN6390 replicates and induces apoptosis in a pulmonary epithelial cell line derived from a cystic fibrosis patient. Infect. Immun. 68:5385‐5392.
   Lowy, F. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339:520‐532.
   Piqueras, B., Autran, B., Debre, P., and Gorochov, G. 1996. Detection of apoptosis at the single cell level by direct incorporation of fluorescein‐dUTP in DNA strand breaks. Biotechniques 20:634‐640.
   Qazi, S.N., Harrison, S.E., Self, T., Williams, P., and Hill, P.J. 2004. Real‐time monitoring of intracellular Staphylococcus aureus replication. J. Bacteriol. 186:1065‐1077.
   Strober, 1997. Trypan blue test of cell viability. In Current Protocols in Immunology (J.E. Coligan, B.E. Bierer, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.3B.1‐A.3B.2. John Wiley & Sons, Hoboken, N.J.
   Wijsman, J.H., Jonker, R.R., Keijzer, R., Van de Velde, C.J.H., Cornelisse, C.J., and Van Dierendonck, J.H. 1993. A new method to detect apoptosis in paraffin sections: In situ end‐labeling of fragmented DNA. J. Histochem. Cytochem. 41:7‐12.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library