Name: Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria
Uploaded: 2023-04-30T15:01:28
Description: Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial ...

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1. Cell inoculation
<ol>
	<li>Begin with a culture of A549 human epithelial cells in Dulbecco&#39;s modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics.</li>
	<li>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.</li>
	<li>Remove and discard the spent cell culture medium from the 24-well plate.</li>
	<li>Add 500 &#181;L of the bacterial suspension of Staphylococcus aureus prepared in DMEM without phenol red, for inoculation to each well with 100% confluent cells.</li>
	<li>Incubate the cells for 2 h at 36 &#177; 1 &#176;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.</li>
</ol>
2. Quantification of intracellular bacteria with improved enzyme protection assay (iEPA)
<ol>
	<li>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.</li>
	<li>Prepare a lysostaphin stock solution at 10 mg/mL in acetate buffer and aliquot 25 &#181;L into cryovials. Store at -80 &#176;C for up to 6 months.</li>
	<li>Prepare 250 &#181;L of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 &#181;L of the lysostaphin stock solution (10 mg/mL) and 225 &#181;L of 0.1 M Tris-HCl. Store at 4 &#176;C for up to 48 h.</li>
	<li>Prepare 6.25 mL of complete infection medium supplemented with lysostaphin by adding 6 mL of complete infection medium to 250 &#181;L of the lysostaphin working solution.</li>
	<li>Add 250 &#181;L of complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swivelling the plate by hand.</li>
	<li>Incubate the cells for 1 h at 36 &#177; 1 &#176;C in 5% CO2&#160;to let the lysostaphin kill the extracellular bacteria.</li>
	<li>At the end of the incubation time, add 10 &#181;L of proteinase K at 20 mg/mL into each well to inactivate the lysostaphin.</li>
	<li>Incubate the cells for 2 min at room temperature.</li>
	<li>Add 250 &#181;L of 4x lysis buffer to lyse the cells by osmotic shock.</li>
	<li>Incubate the cells for 10 min at 36 &#177; 1 &#176;C.</li>
	<li>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.</li>
	<li>Use an automatic spiral plater to determine the&#160;S. aureus&#160;load of each well.</li>
	<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
	<li>The next day, count the number of colonies with a colony counter to calculate the intracellular&#160;S. aureus&#160;load of each well.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plate</td>
			<td>CORNING-FALCON</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cell line</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate buffer solution pH 4.6</td>
			<td>Fluka</td>
			<td>31048</td>
			<td>Used to prepare lysostpahine stock solution at 10 mg/mL in 20 mM sodium acetate.</td>
		</tr>
		<tr>
			<td>AMBICIN (Recombinant lysostaphin)</td>
			<td>AMBI</td>
			<td>LSPN-50</td>
			<td>Lyophilized recominant lysostaphin. Freeze at -80 &#176;C for long-term storage.</td>
		</tr>
		<tr>
			<td>COS - Colombia agar + 5% sheep blood</td>
			<td>Biomerieux</td>
			<td>43049</td>
			<td>Any agar plate suitable for growing staphylococci can be used instead.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium, high glucose with phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium, high glucose without phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>D1145</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate-buffered Saline with MgCl2&#160;and CaCl2, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate-buffered Saline, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Easyspiral dilute</td>
			<td>Interscience</td>
			<td>414000</td>
			<td>Automatic diluter and spiral plater</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant 20 mg/mL</td>
			<td>Eurobio</td>
			<td>GEXPRK01-B5</td>
			<td>&#62; 30 U/mg, lot 901727</td>
		</tr>
		<tr>
			<td>Scan 4000</td>
			<td>Interscience</td>
			<td>438000</td>
			<td>Automatic colony counter</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>OTEC</td>
			<td>600500</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Biorad</td>
			<td>1450102</td>
			<td>Automatic cell counter</td>
		</tr>
		<tr>
			<td>Tris 1 M pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9855G</td>
			<td>Used to prepare lysostaphine working solution at 1 mg/mL in 0.1 M Tris-HCl.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin - EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>0.05% porcine trypsin and 0.02% EDTA in Hanks&#8242; Balanced Salt Solution with phenol red</td>
		</tr>
		<tr>
			<td>Wide-field fluorescence microscope</td>
			<td>Nikon</td>
			<td>Ti2</td>
			<td></td>
		</tr>
	</tbody>
</table>

lysostaphin-based enzyme protection assay: an in vitro method to quantify intracellular staphylococcus aureus load by enzyme-mediated selective killing of extracellular bacteria

Source: Rigaill, J. et al. <a href="https://www.jove.com/v/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds.</a> 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.

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

Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial ...

Begin with a monolayer of epithelial cells in a suitable culture medium. These cells express integrins - membrane-spanning surface receptors. The culture media contains epithelial cell-secreted fibronectin - a glycoprotein.
Add a suspension of Staphylococcus aureus - a gram-positive pathogenic bacteria - to the culture plate. These bacteria contain fibronectin-binding proteins, or FnBPs, anchored in their cell wall.
Incubate the plate to initiate bacterial infection, wherein the fibronectin binds to the bacteria&#39;s FnBP and the integrins on the epithelial host cells. This tripartite FnBP-fibronectin-integrin interaction mediates bacterial internalization in the host.
Treat the cells with lysostaphin - an endopeptidase enzyme - and incubate. Lysostaphin selectively enters the cell walls of extracellular bacteria. It cleaves the pentaglycine bridges in the peptidoglycans, thereby lysing them while the intracellular bacteria remain protected from lysostaphin.
Supplement the culture with a proteolytic enzyme to deactivate the remaining lysostaphin, preventing it from killing the intracellular bacteria in the steps.
Add a lysis buffer to the culture, which ruptures the host epithelial cells and releases intracellular bacteria in the cell lysate. Subsequently, plate the cell lysate containing bacteria on a suitable culture plate and incubate.
The bacteria grow and multiply to form bacterial colonies, which can be analyzed to estimate the extent of bacterial internalization in the host cells.&#160;

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162 Views. 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. 

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

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Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

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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

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