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.
| Errataetall: | |
|---|---|
| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2023 |
|---|---|
| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2023 |
|---|---|
| Enthalten in: |
Journal of visualized experiments : JoVE - (2023), 202 vom: 08. Dez. |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Jayaswal, Praapti [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
|---|
| Anmerkungen: |
Date Completed 26.12.2023 Date Revised 26.02.2024 published: Electronic ErratumIn: J Vis Exp. 2024 Feb 1;(204):. - PMID 38300134 Citation Status MEDLINE |
|---|
| doi: |
10.3791/65138 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM36636040X |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM36636040X | ||
| 003 | DE-627 | ||
| 005 | 20240229163940.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231227s2023 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.3791/65138 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1308.xml |
| 035 | |a (DE-627)NLM36636040X | ||
| 035 | |a (NLM)38145372 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Jayaswal, Praapti |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria |
| 264 | 1 | |c 2023 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 26.12.2023 | ||
| 500 | |a Date Revised 26.02.2024 | ||
| 500 | |a published: Electronic | ||
| 500 | |a ErratumIn: J Vis Exp. 2024 Feb 1;(204):. - PMID 38300134 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Video-Audio Media | |
| 650 | 7 | |a Bacterial Proteins |2 NLM | |
| 700 | 1 | |a Ilyas, Mohd |e verfasserin |4 aut | |
| 700 | 1 | |a Singh, Kuljit |e verfasserin |4 aut | |
| 700 | 1 | |a Kumar, Saurabh |e verfasserin |4 aut | |
| 700 | 1 | |a Sisodiya, Lovely |e verfasserin |4 aut | |
| 700 | 1 | |a Jain, Sapna |e verfasserin |4 aut | |
| 700 | 1 | |a Mahlawat, Rahul |e verfasserin |4 aut | |
| 700 | 1 | |a Sharma, Nishant |e verfasserin |4 aut | |
| 700 | 1 | |a Gupta, Vishal |e verfasserin |4 aut | |
| 700 | 1 | |a Atmakuri, Krishnamohan |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Journal of visualized experiments : JoVE |d 2006 |g (2023), 202 vom: 08. Dez. |w (DE-627)NLM181595761 |x 1940-087X |7 nnns |
| 773 | 1 | 8 | |g year:2023 |g number:202 |g day:08 |g month:12 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.3791/65138 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |j 2023 |e 202 |b 08 |c 12 | ||