Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria

Published: April 30, 2023

Abstract

Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds. J. Vis. Exp. (2021).

In this video, we demonstrate a lysostaphin-based in vitro enzyme protection assay to quantify the Staphylococcus aureus internalization in A549 cells. The assay helps in qualitative, quantitative, and characterization studies of the intracellular bacterial load in the host cells.

Protocol

1. Cell inoculation

  1. Begin with a culture of A549 human epithelial cells in Dulbecco's modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics.
  2. Observe every well of the 24-well plate by low magnification microscopy to ensure that the human epithelial cells are healthy and growing as expected.
  3. Remove and discard the spent cell culture medium from the 24-well plate.
  4. Add 500 µL of the bacterial suspension of Staphylococcus aureus prepared in DMEM without phenol red, for inoculation to each well with 100% confluent cells.
  5. Incubate the cells for 2 h at 36 ± 1 °C and 5% CO2.
    NOTE: it is recommended to use three wells of the plate for each condition to be tested (triplicate) and to perform at least three independent experiments. The delay of incubation can be adapted according to the experimental aim.

2. Quantification of intracellular bacteria with improved enzyme protection assay (iEPA)

  1. Prepare 7 mL of 4x lysis buffer with 3.5 mL of 2% Triton X-100 in sterile water and 3.5 mL of trypsin-EDTA.
  2. Prepare a lysostaphin stock solution at 10 mg/mL in acetate buffer and aliquot 25 µL into cryovials. Store at -80 °C for up to 6 months.
  3. Prepare 250 µL of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 µL of the lysostaphin stock solution (10 mg/mL) and 225 µL of 0.1 M Tris-HCl. Store at 4 °C for up to 48 h.
  4. Prepare 6.25 mL of complete infection medium supplemented with lysostaphin by adding 6 mL of complete infection medium to 250 µL of the lysostaphin working solution.
  5. Add 250 µL of complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swivelling the plate by hand.
  6. Incubate the cells for 1 h at 36 ± 1 °C in 5% CO2 to let the lysostaphin kill the extracellular bacteria.
  7. At the end of the incubation time, add 10 µL of proteinase K at 20 mg/mL into each well to inactivate the lysostaphin.
  8. Incubate the cells for 2 min at room temperature.
  9. Add 250 µL of 4x lysis buffer to lyse the cells by osmotic shock.
  10. Incubate the cells for 10 min at 36 ± 1 °C.
  11. Mix thoroughly by pipetting up and down ten times all over the bottom of the well to ensure that the cells are fully lysed and homogenized.
  12. Use an automatic spiral plater to determine the S. aureus load of each well.
  13. Incubate the agar plates for 18-24 h at 36 ± 1 °C.
  14. The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well.

Divulgations

The authors have nothing to disclose.

Materials

24-well plate CORNING-FALCON 353047
A549 cell line ATCC CCL-185
Acetate buffer solution pH 4.6 Fluka 31048 Used to prepare lysostpahine stock solution at 10 mg/mL in 20 mM sodium acetate.
AMBICIN (Recombinant lysostaphin) AMBI LSPN-50 Lyophilized recominant lysostaphin. Freeze at -80 °C for long-term storage.
COS – Colombia agar + 5% sheep blood Biomerieux 43049 Any agar plate suitable for growing staphylococci can be used instead.
Dulbecco's Modified Eagle's Medium, high glucose with phenol red Sigma-Aldrich D6429
Dulbecco's Modified Eagle's Medium, high glucose without phenol red Sigma-Aldrich D1145
Dulbecco's Phosphate Buffered Saline Sigma-Aldrich D8537
Dulbecco′s Phosphate-buffered Saline with MgCl2 and CaCl2, sterile-filtered Sigma-Aldrich D8662
Dulbecco′s Phosphate-buffered Saline, sterile-filtered Sigma-Aldrich D8537
Easyspiral dilute Interscience 414000 Automatic diluter and spiral plater
Fetal bovine serum Gibco 10270-106
Proteinase K, recombinant 20 mg/mL Eurobio GEXPRK01-B5 > 30 U/mg, lot 901727
Scan 4000 Interscience 438000 Automatic colony counter
Sterile water OTEC 600500
TC20 Automated cell counter Biorad 1450102 Automatic cell counter
Tris 1 M pH 8.0 Invitrogen AM9855G Used to prepare lysostaphine working solution at 1 mg/mL in 0.1 M Tris-HCl.
Triton X-100 Sigma-Aldrich T8787
Trypsin – EDTA solution Sigma-Aldrich T3924 0.05% porcine trypsin and 0.02% EDTA in Hanks′ Balanced Salt Solution with phenol red
Wide-field fluorescence microscope Nikon Ti2

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Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria. J. Vis. Exp. (Pending Publication), e21158, doi: (2023).

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