Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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'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 infection^3,4,5. 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 significance^3,6. 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 component^3,4,7.

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.

Protokół

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 is not feasible, the following procedures need to be performed in a high-containment laboratory.

1. Preparation of crude Mtb EV concentrate

NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see References^11,12. It is recommended that bacterial culture media is free from growth supplements with EV-containing or proteinaceous components, such as Oleic Albumin Dextrose Catalase (OADC), and detergents such as Tween. It is also recommended that the bacterial culture's quality and harvested CFP be screened to ensure limited cell death and lysis^13,14.

1. Prepare a 100 kDa molecular weight cut-off (MWCO) centrifugal filter (see Table of Materials) by adding the full volume capacity of phosphate-buffered saline (1x PBS). Centrifuge for 5 min at 2,800 x g at 4 °C.
   NOTE: If using a filter with no dead stop volume, care must be taken to ensure the sample volume does not reduce below the filter level, resulting in complete filter drying during centrifugation.
2. Discard the flow-through and any remaining PBS before filling the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 x g at 4 °C until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary, adding more CFP to the unit until the entire sample has been reduced sufficiently.
   NOTE: Retain a portion of the 100 kDa flow-through (100F) material for downstream qualification if desired. Store at 4 °C.
3. Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described in 1.2. Repeat this step five times to ensure complete washing and buffer exchange.
4. Recover the 100 kDa concentrated CFP retentate (100R) according to the ultrafiltration device specifications (see Table of Materials). Once the 100R is recovered, wash the filter with a minimal volume of 1x PBS at least three times, and pool the wash with the 100R to maximize the recovery.
5. Quantitate the 100R material with a bicinchoninic assay (BCA) following the manufacturer's instructions (see Table of Materials). To perform the assay, use several dilutions of samples 1:2, 1:5, and 1:10 in PBS. Test the sample in triplicate.
   ​NOTE: Retain a portion of the 100R material for downstream qualification if desired. Store at 4 °C.

2. Size exclusion chromatography for the enrichment of Mtb EVs from CFP

NOTE: The following procedure is specific for using 3 mg of 100R Mtb CFP with SEC column and automatic fraction collector (AFC, see Table of Materials). It can be adapted for other starting concentrations and column types by following the manufacturer's specifications. Additionally, the users are recommended to read and understand the automatic fraction collector user manual.

1. Allow the SEC column to equilibrate to room temperature.
2. Turn on the AFC using the power switch on the back of the tower unit and adjust the settings for the load cell, if necessary, by pressing SETUP on the main menu touchscreen. The load cell must only be calibrated upon first use, after every software update, and if inconsistency in fraction collection volumes is noticed.
3. From the SETUP screen, align the carousel by selecting CAROUSEL > CALIBRATE. Insert the carousel with the 13 small holes facing up into the AFC tower, and adjust the carousel so that the fluid nozzle is directly above the flush position by pressing the - and/or + buttons.
4. Remove the caps from the equilibrated SEC column. Slide the SEC column into the appropriate column mount and carefully install it on the AFC tower. Ensure the radio frequency identification (RFID) tag on the column faces the AFC, and check that the connection between the column and the valve is secure. Place the waste outlet tubing in a collection container.
5. From the SETUP screen, select Collection Schedule and set the count to 13 and the size to 0.5 mL by pressing the - and/or + buttons. Leave the "Buffer Volume" setting as the default of 2.7 mL, and then close the "Collection Schedule" window by pressing X.
6. Select START COLLECTION from the home screen and confirm the collection parameters by selecting YES. Load 13 labeled 1.7 mL microcentrifuge tubes with the lids open and pointing toward the carousel center.
7. Advance the AFC by selecting OK, then mount the column reservoir and advance again by pressing OK. Select the option to flush the column by pressing YES, and add one column volume of 0.2 µm filtered PBS to remove any storage buffer. Once the flush is complete, advance the AFC by pressing OK.
8. Place the carousel cover over the AFC and press OK. Prepare one column volume of 0.2 µm filtered PBS. Use a pipette to remove any excess buffer from the column.
9. Bring 3 mg of 100R sample as quantified in step 1.5 to 500 µL with 1x PBS. Add the 3 mg sample to the top of the column. Advance the AFC and allow the sample to run into the frit. Once the sample has fully entered the column, add the PBS prepared in step 2.6 to the reservoir.
10. Monitor the run of the AFC while it collects first the void volume and then the specified fractions. After the run completes and the carousel returns to waste position, remove the cover, and remove the fraction tubes from the carousel. Store the fractions at 4 °C prior to quantification (step 3) and qualification (step 4).
11. If the column is to be reused, select the option to clean the column by pressing YES; otherwise, press NO. Follow the instructions on the AFC.
    ​NOTE: Once the column is washed and the storage buffer has run through, it can be removed, capped, and stored at 4 °C.

3. Quantification of the Mtb EVs

1. Measure the protein concentration for each fraction using BCA and micro BCA¹².
   1. For 0.5 mL fractions collected from 3 mg of 100R Mtb CFP, use the micro BCA with a 1:3 dilution for fractions numbered 1-7 of each sample in triplicate.
   2. For 0.5 mL fractions numbered 5-13, use the BCA with no dilution for each sample in triplicate.
      NOTE: Overlapping the assays for fractions 5-7 will help ensure all fractions have a readable output.
2. Quantitate the particle concentration for each fraction using nanoparticle tracking analysis (NTA).
   1. Dilute the samples in 1 mL of 1x PBS using the following suggested starting ratios; for fractions 1-3, use 1:100, and then for the remaining fractions, use a 1:10 dilution.
   2. Vortex the diluted solutions and then draw the sample into a 1 mL disposable syringe. Set the syringe in an automatic syringe pump if available.
   3. Set the syringe pump to 30 µL/min and use the video capture settings with a screen gain of 10-12 and a camera level of 10-12. Adjust the focus for each sample.
   4. Collect a minimum of three videos at 30 s each using a constant flow.
   5. Perform the analysis with the software detection threshold set to five.
      ​NOTE: It may not be possible to obtain NTA data for the latest fractions (>7) without sacrificing significant sample volume.

4. Qualification of Mtb EVs

1. Visualize the general protein profile of each fraction using a silver-stained protein gel.
   NOTE: Detailed procedures for SDS-PAGE and silver stain can be found in Reference¹⁵.
   1. Add SDS sample buffer to 10 µL of each fraction and 5 µg of CFP, 100R, and 100F. Boil for 5 min at 100 °C, then load on a 4%-12% Bis-Tris Gel with a molecular weight ladder for polyacrylamide gel electrophoresis (PAGE) (see Table of Materials).
   2. Run the gel using 1x 2-[N-morpholino]ethanesulfonic acid (MES) running buffer (see Table of Materials) and a 200 V current for 35 min.
   3. Remove the gel from the cassette and proceed with silver staining¹⁵.
2. Evaluate the presence or absence of EV markers in each fraction by Western blot.
   NOTE: Detailed procedures for Western blotting can be found in Reference¹⁵.
   1. Run an SDS-PAGE gel as described in steps 4.1.1-4.1.2.
   2. Remove the gel from the cassette and transfer the resolved proteins to a 0.2 µm nitrocellulose membrane (see Table of Materials) by applying a 50 V current for a minimum of 1 h.
   3. Perform Western blot analysis to evaluate proteins of interest. Primary antibodies against LpqH, lipoarabinomannan (LAM), and GroES (see Table of Materials) are recommended.
   4. Pool fractions based on the presence or absence of markers as applicable for the downstream application.
3. Confirm the presence of intact vesicles by transmission electron microscopy (TEM).
   1. Fix 15 µL of the vesicle sample by adding 15 µL of 4% EM grade paraformaldehyde and store at 4 °C overnight.
   2. Prepare a 200 mesh formvar-carbon coated copper TEM grid by cleaning the surface and making a hydrophilic substrate.
      NOTE: Plasma cleaning with 95% argon, 5% oxygen, and 30% plasma power for 1 min is recommended.
   3. Drop 10 µL of the fixed sample onto the grid and allow the sample to adhere for 10 min, then blot away the excess liquid with filter paper.
   4. Float the grid on a drop of ultrapure water for 30 s, then blot away excess liquid.
   5. Float the grid on a d% EM grade uranyl acetate drop for 2 min, then blot away the excess liquid.
   6. Allow the grid to air dry completely.
   7. Image using a TEM (see Table of Materials) at 100 kV or similar.
      NOTE: Other EV quantitation and characterization methods should be used based on downstream applications. The Minimal Information for Studies of Extracellular Vesicles¹⁸ should be consulted for guidelines to ensure the rigor and reproducibility of all EV studies.

Wyniki

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...

Dyskusje

Mycobacterium tuberculosis extracellular vesicles are highly antigenic reservoirs, which present them as an attractive avenue for developing diagnostic tools and future vaccines^4,19,20. 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...

Materiały

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