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

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.

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