Name: mycobacterium tuberculosis extracellular vesicle enrichment through size exclusion chromatography
Uploaded: 2022-05-19T15:00:00
Description: Watch this Scientific Journal Video about Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography at JoVE.com

We would like to acknowledge support from the College of Veterinary Medicine and Biomedical Sciences Experiential Award and College Research Council Shared Research Program to NKG and funding by ATCC (award # 2016-0550-0002) to KMD. We would also like to acknowledge Anne Simpson for technical support and BEI Resources, NIAID, NIH for the following reagents: Monoclonal Anti-Mycobacterium tuberculosis LpqH (Gene Rv3763), IT-54 (produced in vitro), NR-13792, Monoclonal Anti-Mycobacterium tuberculosis GroES (Gene Rv3418c), Clone IT-3 (SA-12) (produced in vitro), NR-49223, and Monoclonal Anti-Mycobacterium tuberculosis LAM, Clone CS-35 (produced in vitro), NR-13811.

The Colorado State University Institutional Biosafety Committee approved the present study (19-046B). Cultivation of Mycobacterium tuberculosis and harvesting of EV-rich culture supernatants were performed by trained personnel in a high-containment laboratory. The materials were moved out of the high-containment area after a valid inactivation method was performed, confirmed, and approved by institutional biosafety policies. While replicating the protocol, if validated inactivation or sterile filtration method i...

<ol>
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Mycobacterium tuberculosis extracellular vesicles are highly antigenic reservoirs, which present them as an attractive avenue for developing diagnostic tools and future vaccines4,19,20. Historically, density gradient ultracentrifugation has been used to separate Mtb EVs from other soluble, secreted material8. While this process is effective, it is also time-consuming, technically challenging, and...

Extracellular vesicle (EV) release by pathogens may be the key to unlocking new technologies to control infectious diseases1. Mycobacterium tuberculosis (Mtb) is a pathogen of high consequence, infecting approximately one-third of the world&#39;s population and claiming the lives of millions of people each year2. EV production by Mtb is well documented yet elusive in the biogenesis and varied roles (i.e., immunostimulatory, immunosuppressive, iron and nutrient acquisition) of these EVs in the context of infection3,4,5. Efforts to understand the composition of Mtb EVs revealed 50-150 nm lipid membrane-enclosed spheres derived from the plasma membrane containing lipids and proteins of immunological significance3,6. Investigation of the role of Mtb EVs in bacterial physiology has revealed the importance of bacterial EV modulation in response to environmental stress for survival5. Host-pathogen interaction studies have been more complicated to interpret, but evidence indicates that Mtb EVs can influence the immune response of the host and may potentially serve as an effective vaccination component3,4,7.
Most studies of Mtb EVs thus far have relied on density gradient ultracentrifugation for vesicle enrichment8. This has been effective for small-scale studies; however, this technique has several technical and logistical challenges. Alternate workflows couple multistep centrifugation, for the removal of whole cells and large debris, with a final ultracentrifugation step to pellet EVs. This methodology can vary in efficiency, and often results in low yield and co-purification of soluble non-vesicle associated biomolecules while also impacting vesicle integrity9. Additionally, this process is time-consuming, manually intensive, and very limited in throughput due to equipment constraints.
The present protocol describes an alternative technique to density gradient ultracentrifugation: size exclusion chromatography (SEC). This method has been demonstrated for environmental mycobacteria, and in the current work, it has been extrapolated to Mtb10. A commercially available column and automatic fraction collector can improve consistency in vesical preparation and reduce the necessity for specific, expensive equipment. It is also possible to complete this protocol in a fraction of the time compared to density gradient ultracentrifugation, increasing the throughput. This technique is less technically challenging, making it easier to master, and can increase inter/intra-laboratory reproducibility. Finally, SEC has high separation efficiency and is gentle, preserving the integrity of the vesicles.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20x MES SDS Running Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>15705-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>IZON Science</td>
			<td>AFC-V1-USD</td>
			<td></td>
		</tr>
		<tr>
			<td>BenchMark Pre-stained Protein Ladder</td>
			<td>Invitrogen</td>
			<td>10748010</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra 6R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centricon Plus - 70 Centrifugal filter, 100 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC710008</td>
			<td>Ultrafiltration device used in step 1.1</td>
		</tr>
		<tr>
			<td>Electroblotting System</td>
			<td>ThermoFisher Scientific</td>
			<td>09-528-135</td>
			<td></td>
		</tr>
		<tr>
			<td>EM Grade Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar/Carbon 200 mesh Cu Grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>FCF200H-Cu-TA</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L (Alkaline Phosphatase), whole molecule, 1 mL</td>
			<td>AbCam</td>
			<td>ab6790</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>JEM-1400 Transmission Electron Microscope</td>
			<td>JOEL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BIOTEK</td>
			<td>Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Mycobacterium tuberculosis GroES (Gene Rv3814c)</td>
			<td>BEI Resources</td>
			<td>NR-49223</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Mycobacterium tuberculosis LpqH (Gene Rv3763)</td>
			<td>BEI Resources</td>
			<td>NR-13792</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Monocolonal Anti-Mycobacterium tuberculosis LAM, Clone CS-35</td>
			<td>BEI Resources</td>
			<td>NR-13811</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>NanoClean 1070</td>
			<td>Fischione Instruments</td>
			<td></td>
			<td>For plasma cleaning of the TEM grid</td>
		</tr>
		<tr>
			<td>Nanosight equipped with syringe pump and computer with NanoSight NTA software</td>
			<td>Malvern Panalytical</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane, Roll, 0.2 &#956;m</td>
			<td>BioRad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>ThermoFisher Scientific</td>
			<td>NP0323BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline, 1X without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BioRad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>qEV Original 35 nm 5/pk</td>
			<td>IZON Science</td>
			<td>SP5-USD</td>
			<td>SEC column</td>
		</tr>
		<tr>
			<td>SDS sample buffer</td>
			<td>Boster</td>
			<td>AR1112</td>
			<td>In-house recipe used in this procedure, however this product is equivalent</td>
		</tr>
		<tr>
			<td>SDS-PAGE gel chamber</td>
			<td>ThermoFisher Scientific</td>
			<td>EI0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmafast BCIP/NBT</td>
			<td>Millipore Sigma</td>
			<td>B5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Stain Plus Kit</td>
			<td>BioRad</td>
			<td>1610449</td>
			<td>In-house protocol used in this procedure, however this kit is equivalent</td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
	</tbody>
</table>

mycobacterium tuberculosis extracellular vesicle enrichment through size exclusion chromatography

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. Specifically, Mycobacterium tuberculosis (Mtb) EVs play a role in the host-pathogen interaction and response to environmental stress. Mtb EVs are also highly antigenic and show potential as vaccine components. The most common method for purifying Mtb EVs is density gradient ultracentrifugation. This process has several limitations, including low throughput, low yield, reliance on expensive equipment, technical challenges, and it can negatively impact the resulting preparation. Size exclusion chromatography (SEC) is a gentler alternative method that combats many of the limitations of ultracentrifugation. This protocol demonstrates that SEC is effective for Mtb EV enrichment and produces high-quality Mtb EV preparations of increased yield in a rapid and scalable manner. Additionally, a comparison to density gradient ultracentrifugation by quantification and qualification procedures demonstrates the benefits of SEC. While the evaluation of EV quantity (nanoparticle tracking analysis), phenotype (transmission electron microscopy), and content (Western blotting) is tailored to Mtb EVs, the workflow provided can be applied to other mycobacteria.

Culture filtrate protein (CFP) from Mycobacterium tuberculosis (Mtb) was concentrated, quantified, and then 3 mg of material was applied to a size exclusion chromatography (SEC) column. The protein and particle concentrations were enumerated by BCA and NTA, respectively. Expected ranges for protein and particle recovery plus the exact values obtained for these results are reported in Table 1. Values much higher than these ranges may indicate contamination or column integrity issues. Values signi...

Watch this Scientific Journal Video about Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography at JoVE.com

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

This protocol describes size exclusion chromatography, a facile and reproducible technique for enriching Mycobacterium tuberculosis extracellular vesicles from culture supernatants.

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. ...

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