Name: enrichment of native and recombinant extracellular vesicles of mycobacteria
Uploaded: 2023-12-08T12:00:00
Description: Watch this Scientific Journal Video about Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria at JoVE.com

The authors sincerely thank Prof. Sarah M. Fortune for kindly sharing M. smegmatis mc2155 stock. They also acknowledge Servier Medical Art (smart.servier.com) for providing some basic elements for Figure 1. They sincerely acknowledge the support of the rest of the lab members for their patient adjustments during the long use of the incubator shakers, centrifuges, and ultracentrifuges for mEV enrichment. They also acknowledge Mr. Surjeet Yadav, the laboratory assistant, for always making sure the necessary glassware and consumables were always available and handy. Lastly, they acknowledge the administrative, the purchase, and the finance teams of THSTI for their constant support and help in the seamless execution of the project.

1. Growth conditions of Mycobacterium smegmatis, Escherichia coli, and their derivatives
<ol>
	<li>Media
	<ol>
		<li>Middlebrook 7H9 liquid broth
		<ol>
			<li>Prepare 20% Tween-80 stock solution by pre-warming the required volume of double-distilled water (ddw) in a glass beaker to ~45-50 oC in a microwave, add the required volume of Tween-80 using an appropriate measuring cylinder, and stir continuously on a small magnetic stirrer to bri...

Since developing a novel TB vaccine that is superior to and can replace BCG remains a formidable challenge, as an alternative, several groups are pursuing the discovery of different subunit TB vaccines that can boost BCG&#39;s potency and extend its protective duration48,49. Given the increasing attention to bacterial EVs (bEVs) as potential subunits and as natural adjuvants50,51, consistent enrichment of...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65138">Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria</a>. The Authors section was updated from:
Praapti Jayaswal1 
Mohd Ilyas1 
Kuljit Singh1,2 
Saurabh Kumar1,3 
Lovely Sisodiya1 
Sapna Jain1 
Rahul Mahlawat1 
Nishant Sharma1 
Vishal Gupta1 
Krishnamohan Atmakuri1 
1Bacterial Pathogenesis Laboratory, Infectious Diseases and Immunology Group, Translational Health Science and Technology Institute, NCR Biotech Science Cluster 
2Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine 
3ICAR-Research Complex for Eastern Region 
4Public Health Research Institute, Rutgers University
to:
Praapti Jayaswal1 
Mohd Ilyas1 
Kuljit Singh1,2 
Saurabh Kumar1,3 
Lovely Sisodiya1 
Sapna Jain1 
Rahul Mahlawat1 
Nishant Sharma1,4 
Vishal Gupta1 
Krishnamohan Atmakuri1 
1Bacterial Pathogenesis Laboratory, Infectious Diseases and Immunology Group, Translational Health Science and Technology Institute, NCR Biotech Science Cluster 
2Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine 
3ICAR-Research Complex for Eastern Region 
4Public Health Research Institute, Rutgers University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65138">Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria</a>. The Authors section was updated.

Erratum: Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Despite the development and administration of a wide range of vaccines against infectious diseases, even to this day, ~30% of all human deaths still occur from communicable diseases1. Before the advent of the Tuberculosis (TB) vaccine - Bacillus Calmette Guerin (BCG) - TB was the number one killer (~10,000 to 15,000/100,000 population)2. With the administration of BCG and easy access to first and second-line anti-TB drugs, by 2022, TB-related deaths have dramatically dropped to ~1 million/year by 2022 (i.e., ~15-20/100,000 population1). However, in TB endemic populations of the world, TB-related deaths continue to stand at ~100-550/100,000 population1. While experts recognize several reasons leading to these skewed numbers, BCG-mediated protection not lasting for even the first decade of life appears to be the prominent reason3,4,5,6,7. Consequently, given the renewed &#39;Sustainable Development Goals&#39; of the UN and the &#39;End TB Strategy&#39;, of WHO, there is a concerted global effort to develop a much superior vaccine alternative to BCG that perhaps provides lifelong protection from TB.

Towards that objective, several groups are currently evaluating modified/recombinant BCG strains, non-pathogenic and attenuated mycobacterial species other than BCG, and subunit candidates8,9,10,11,12,13,14,15,16,17,18. Typically, subunit vaccines are liposomes selectively loaded with few purified (~1-6) full-length or truncated immunogenic proteins of the pathogen. However, because of their spurious folding into non-native conformations and/or random non-functional interactions between the loaded proteins, subunits often lack native and germane epitopes and hence, fail to sufficiently prime the immune system14,19,20.

Consequently, extracellular vesicles (EVs) of bacteria have picked up pace as a promising alternative21,22,23,24,25,26. Typically, bacterial EVs (bEVs) contain a subset of their cellular components, including some portions of nucleic acids, lipids, and hundreds of metabolites and proteins27,28. Unlike liposomes where a few purified proteins are artificially loaded, bEVs contain hundreds of naturally-loaded, natively-folded proteins with a better propensity to prime the immune system, especially without the boost/aid of adjuvants and Toll-like receptor (TLR) agonists27,28,29. It is in this line of research that we and others have explored the utility of mycobacterial EVs as potential subunit boosters to BCG30. Despite concerns that bEVs lack uniform antigen loads, EVs from attenuated Neisseria meningitidis have successfully protected humans against serogroup B meningococcus31,32.
At least theoretically, the best EVs that could boost BCG well are the EVs enriched from pathogenic bacteria. However, enriching EVs generated by pathogenic mycobacterium is expensive, time-consuming, and risky. Additionally, pathogen-generated EVs may be more virulent than protective. Given the potential risks, here, we report a well-tested protocol for the enrichment of EVs generated by axenically grown Msm, an avirulent mycobacterium.
However, despite encoding several pathogen protein orthologs, avirulent mycobacteria lack several vaccine antigens/pathogenic protein epitopes necessary to sufficiently prime the immune system towards protection33. Therefore, we also explored constructing and enriching recombinant EVs of Msm through molecular engineering, such that a significant portion of any pathogenic protein of interest expressed and translated in Msm, must reach its EVs. We hypothesized that one or more of the top 10 abundant proteins of Msm EVs when fused to the protein of interest will aid in such translocation.
While we were beginning to standardize the enrichment of mycobacterial EVs (mEVs) in our laboratory, in 2011, Prados-Rosales et al. first reported the visualization and enrichment of mEVs in vitro30. Later, in 2014, the same group published a modified version of their 2011 method34. In 2015, Lee et al. also reported an independently standardized method for mEV enrichment again from axenic cultures of mycobacteria35. Combining both protocols34,35 and incorporating a few of our modifications after thorough standardization, we describe here a protocol that helps routinely enrich mEVs from axenic cultures of mycobacteria36.
Here, we particularly detail the enrichment of Msm-specific EVs, which is an extension of a published protocol36 for the enrichment of mycobacterial EVs in general. We also detail how to construct recombinant mEVs (R-mEVs) that contain the mCherry protein (as a red fluorescent reporter) and EsxA (Esat-6)37,38,39 a predominant immunogen and a potential subunit vaccinogen of Mycobacterium tuberculosis. The protocol for enriching the R-mEVs remains identical to the one we have described for enriching native EVs from Msm.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A2 type Biosafety Cabinet</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>1300 series</td>
		</tr>
		<tr>
			<td>Bench top Centrifuge</td>
			<td>Eppendorf, USA</td>
			<td></td>
			<td>5810 R</td>
		</tr>
		<tr>
			<td>BstB1, HindIII, HpaI</td>
			<td>NEB, USA</td>
			<td></td>
			<td>NEB</td>
		</tr>
		<tr>
			<td>Cell densitometer</td>
			<td>GE Healthcare, USA</td>
			<td></td>
			<td>Ultraspec 10</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Dibasic Potassium Phosphate</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Double Distilled Water</td>
			<td>Merck, USA</td>
			<td></td>
			<td>~18.2 MW/cm @ 25 oC</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>Bio-Rad, USA</td>
			<td></td>
			<td>2 mm</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-Rad, USA</td>
			<td></td>
			<td>Electroporator</td>
		</tr>
		<tr>
			<td>EsxA-specific Ab</td>
			<td>Abcam, UK</td>
			<td></td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr>
			<td>Ferric Ammonium Citrate</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Floor model centrifuge</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>Sorvall RC6 plus</td>
		</tr>
		<tr>
			<td>Glassware</td>
			<td>Borosil, INDIA</td>
			<td></td>
			<td>1 L Erlenmeyer flasks</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>HEPES and Sodium Chloride</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Incubator shakers</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>MaxQ 6000 &#38; 8000</td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth and Agar, Miller</td>
			<td>Hi Media, INDIA</td>
			<td></td>
			<td>Hi Media</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Tarsons, INDIA</td>
			<td></td>
			<td>Tarsons</td>
		</tr>
		<tr>
			<td>mCherry-specific Ab</td>
			<td>Abcam, UK</td>
			<td></td>
			<td>Rabbit monoclonal</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>LG, INDIA</td>
			<td></td>
			<td>MC3286BLT</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 Broth</td>
			<td>BD, USA</td>
			<td></td>
			<td>Difco Middlebrook 7H9 Broth</td>
		</tr>
		<tr>
			<td>Middlebrook ADC enrichment</td>
			<td>BD, USA</td>
			<td></td>
			<td>BBL Middlebrook ADC enrichment</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>Spectronic 200 UV-Vis</td>
		</tr>
		<tr>
			<td>NEB5a</td>
			<td>NEB, USA</td>
			<td></td>
			<td>a derivative of DH5a</td>
		</tr>
		<tr>
			<td>Optiprep (Iodixanol)</td>
			<td>Merck, USA</td>
			<td></td>
			<td>Available as 60% stock solution (in water)</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Hi Media, INDIA</td>
			<td></td>
			<td>Hi Media</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo, USA</td>
			<td></td>
			<td>Mettler Toledo</td>
		</tr>
		<tr>
			<td>Plasmid DNA mini kit</td>
			<td>Hi Media, INDIA</td>
			<td></td>
			<td>Hi Media</td>
		</tr>
		<tr>
			<td>Plate incubator</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>New Series</td>
		</tr>
		<tr>
			<td>Plasmid pMV261</td>
			<td>Addgene, USA * 
			*The&#160;&#160; plasmid&#160;&#160; is&#160;&#160; no&#160;&#160; more available in this plasmid bank</td>
			<td></td>
			<td>Shuttle vector</td>
		</tr>
		<tr>
			<td>Proof-reading DNA Polymerase</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>Phusion DNA Plus Polymerase</td>
		</tr>
		<tr>
			<td>Q5 Proof-reading DNA Polymerase</td>
			<td>NEB, USA</td>
			<td></td>
			<td>NEB</td>
		</tr>
		<tr>
			<td>Refrigerated circulating water bath</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>R20</td>
		</tr>
		<tr>
			<td>Middlebrock 7H11 Agar base</td>
			<td>BD, USA</td>
			<td></td>
			<td>BBL Seven H11 Agar base</td>
		</tr>
		<tr>
			<td>SOC broth</td>
			<td>Hi Media, INDIA</td>
			<td></td>
			<td>Hi Media</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB, USA</td>
			<td></td>
			<td>NEB</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter, USA</td>
			<td></td>
			<td>Optima L100K</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes - 14 mL</td>
			<td>Beckman Coulter, USA</td>
			<td></td>
			<td>Polyallomer type &#8211; ultra clear type in SW40Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes - 38 mL</td>
			<td>Beckman Coulter, USA</td>
			<td></td>
			<td>Polypropylene type&#8211; cloudy type for SW28 rotor</td>
		</tr>
		<tr>
			<td>Ultrasonics cleaning waterbath sonicator</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>Sonicator - bench top model</td>
		</tr>
		<tr>
			<td>0.22 &#181;m Disposable filters</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>Nunc-Nalgene</td>
		</tr>
		<tr>
			<td>30-kDa Centricon concentrators</td>
			<td>Merck, USA</td>
			<td></td>
			<td>Amicon Ultra centrifugal filters - Millipore</td>
		</tr>
		<tr>
			<td>3X FLAG antibody</td>
			<td>Sigma-Aldrich, Merck, USA</td>
			<td></td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr>
			<td>400 mL Centrifuge bottles</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>Nunc-Nalgene</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tubes</td>
			<td>Corning, USA</td>
			<td></td>
			<td>Sterile, pre-packed</td>
		</tr>
		<tr>
			<td>Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strain</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>NEB, USA</td>
			<td></td>
			<td>NEB 5-alpha (a derivative of DH5&#945;).</td>
		</tr>
		<tr>
			<td>Msm expressing cfp29::mCherry</td>
			<td>This study</td>
			<td></td>
			<td>MC2&#160;155</td>
		</tr>
		<tr>
			<td>Msm expressing cfp29::esxA</td>
			<td>This study</td>
			<td></td>
			<td>MC2&#160;155</td>
		</tr>
		<tr>
			<td>Msm expressing cfp29::esxA::3X FLAG</td>
			<td>This study</td>
			<td></td>
			<td>MC2&#160;155</td>
		</tr>
		<tr>
			<td>Mycobacterium smegmatis (Msm)</td>
			<td>Prof. Sarah M. Fortune, Harvard Univ, USA</td>
			<td></td>
			<td>&#160;MC2&#160;155</td>
		</tr>
	</tbody>
</table>

enrichment of native and recombinant extracellular vesicles of mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

We use M. smegmatis (Msm) as the model mycobacterium to demonstrate the enrichment of both native and recombinant mEVs (R-mEVs). This schematically summarized mEVs enrichment protocol (Figure 1) also works for the enrichment of R-mEVs of Msm and native EVs of Mtb (with minor modifications as in protocol notes of 1.2). Visualization of the enriched mEVs requires negatively staining them under a transmission electron microscope36 (Figure 2A...

Watch this Scientific Journal Video about Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria at JoVE.com

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Enriching mycobacterial EVs helps delineate their contents, decipher their factions, and dates designing ways to construct and generate recombinant EVs that mainly exploit vaccine candidates. Six series generate from different locations of the bacterial surface. They do not have conserved markers.Hence, any affinity-based approaches can enrich only a subset of the EVs. In contrast, our method captures all generated mycobacteria EVs. Demonstrating the procedure will be Mohd Ilyas, a PHD student from my laboratory.To begin, add one milliliter of glycerol stock of wild-type or recombinant Mycobacterium smegmatis to a 50-milliliter centrifuge tube containing 10 milliliters of pre-warmed 7H9 broth. Close the lid and swirl the tube before incubating the culture overnight at 37 degrees Celsius in between 200 to 220 RPM. On the next day, when the optical density at 600 nanometers reaches approximately one, centrifuge the culture at 3, 200 G for 10 minutes at room temperature and discard the supernatant into a liquid discard container.Add one milliliter of pre-warmed Sautons media and gently resuspend to obtain a uniform suspension. Make up the volume to 10 milliliters using Sautons media. Centrifuge again and discard the supernatant.After washing, resuspend the bacterial cells. in 20 milliliters of pre-warmed Sautons media and measure the optical density at 600 nanometers. Inoculate the required volume of bacterial suspension into a one to two liter Erlenmeyer flask containing 330 milliliters of Sautons media and incubate at 37 degrees Celsius in 200 RPM until the optical density reaches 0.3.On the next day, wash the cells once as previously demonstrated and resuspend the pellet in 330 milliliters of Sautons containing 1/10 Tween 80. Distribute 50 milliliters of the cell suspension into six one-liter flasks, each containing 280 milliliters of pre-warmed Sautons containing 1/10 Tween 80. Incubate the cultures at 37 degrees Celsius in 200 RPM until the optical density reaches two to 2.5.Add two liters of mid-exponential stage cultures to six 400-milliliter bottles and centrifuge at 8, 000 G for 20 minutes at four degrees Celsius. Collect the supernatant and pre-chilled autoclaved flasks or beakers and store an aliquot of the pellet for analytical procedures. The pellet is colored if the EVs are recombinant for mCherry and brown if they are wild-type or recombinant for non-fluorescent proteins.Filter the culture supernatant through a 0.45-micron disposal filter, followed by filtration through a 0.22-micron filter to remove all traces of bacteria. Next, pre-wash the 30-kilodalton membrane concentrators with 15 milliliters of double-distilled water at 4, 000 G for 20 minutes at four degrees Celsius. Then wash with 15 milliliters of pre-filtered cold Sautons media using the same conditions to remove all traces of chemicals.After washing, add 15 milliliters of filtered cultured supernatant into a concentrator and concentrate at 4, 000 G for 20 minutes at four degrees Celsius. Transfer the concentrate to a cold 40-milliliter Oak Ridge centrifuge tube or a 50-milliliter fresh Falcon centrifuge tube. Once the entire two liters of culture filtrate is concentrated, transfer the concentrate into a double distilled, washed, and pre-chilled 50-milliliter polypropylene centrifuge tube and subjected to a two-step centrifugation.First at 4, 000 G, and then at 15, 000 G.Transfer the centrifuged concentrate into a pre-chilled 38.5-milliliter ultracentrifuge tube and spin at 100, 000 G for four hours at four degrees Celsius. Collect the supernatant in a pre-chilled 50 milliliter fresh Falcon centrifuge tube. invert the ultracentrifuge tube on a fresh, lint-free absorbent paper to remove traces of the supernatant and resuspend the pellet in 600 microliters of HEPES buffer.Layer the resuspended pellet at the bottom of a 13-milliliter pre-chilled UltraClear polypropylene ultracentrifuge tube and gently mix with four milliliters of inert 60%density gradient iodixanol solution. Then overlay with one milliliter each of 40%30%20%and 10%of iodixanol. Fill the tube by adding four milliliters of 6%iodixanol at the top.After carefully weighing the tube in a glass beaker or a stand, gently transfer it into the swinging buckets and then to the swinging buckets rotor for ultracentrifugation at 141, 000 G for 16 hours at four degrees Celsius. After ultracentrifugation, carefully remove the tube and collect a one milliliter fraction into autoclaved microcentrifuge tubes. TEM analysis showed a mycobacterial EVs are circular and nano-tracking analysis confirmed different sized EVs with diameters ranging between 20 and 250 nanometers.When the N-terminal end of mCherry was translationally fused to the C-terminal end of CFP-29, a portion of enriched mEVs of Msm turned pink, indicating cf-29's ability to carry a foreign protein of interest. CFP-29 was then used to evaluate EsxA delivery into Msm EVs. Mtb's EsxA was observed in Msm EVs in low quantities.CFP-29 EsxA 3X FLAG was more stable and accumulated at higher levels in mEVs. Enriching the EVs from mycobacteria grown under physiological different conditions provides insights into their altered contents and modified functions that dictate sustained infectivity, pathogenesis, and pathogen spread.

enrichment-native-recombinant-extracellular-vesicles

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...