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: 2023-04-30T15:01:28.000+00:00
Duration: 1 min 59 s
Description: Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial ...

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

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

Before cell inoculation, observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected. Then, remove and discard the spent cell culture medium from the 24-well plate, and add 500 microliters of the bacterial suspension to each well containing 100% confluent cells. Incubate the cells for 2 hours at 36 degrees Celsius and 5% carbon dioxide.
To quantify the intracellular bacteria with the improved enzyme protection assay, prepare 4X lysis buffer, 2% Triton X-100, trypsin-EDTA, and lysostaphin stock and working solutions as mentioned in the text manuscript.
Next, prepare 6.25 milliliters of complete infection medium supplemented with lysostaphin, by adding 6 milliliters of complete infection medium to 250 microliters of the lysostaphin working solution. Then, add 250 microliters of the complete infection medium supplemented with lysostaphin into each well, and gently agitate the plate by swiveling the plate by hand.
To allow the lysostaphin to kill the extracellular bacteria, incubate the cells for 1 hour at 36 degrees Celsius and 5% carbon dioxide. Then, inactivate the lysostaphin by adding 10 microliters of proteinase K at 20 micrograms per milliliter into each well, and incubating the cells for 2 minutes at room temperature.
Next, add 250 microliters of 4X lysis buffer to lyse the cells by osmotic shock, and incubate the cells for 10 minutes at 36 degrees Celsius. After the incubation, pipette the cell lysate up and down several times, to ensure that the cells are fully lysed and homogenized.
Then, use an automatic spiral plater to determine the Staphylococcus aureus load of each well, and incubate the agar plates for 18 to 24 hours at 36 degrees Celsius. The next day, count the number of colonies with a colony counter to calculate the intracellular Staphylococcus aureus load of each well.

lysostaphin-based-enzyme-protection-assay-an-vitro-method-to-quantify

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

EoE Sub Articles

eoe sub articles

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>

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.

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;

80 次观看. 基于溶葡萄球菌酶的酶保护测定:一种通过酶介导的细胞外细菌选择性杀伤来量化细胞内金黄色葡萄球菌负荷的体外方法

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

成績單