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Updated: Jun 14, 2025
Isolation and Characterization of Cyanobacterial Extracellular Vesicles
Published on: February 3, 2022
Extraction of Bacterial Membrane Vesicle and Phage Complex by Density Gradient Ultracentrifugation
Shangru Li1,2, Anmin Ren2, Menglu Li1,2
1Shenzhen National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, China.
Abstract:
The bacterial membrane vesicles (MVs) are non-replicative, nanoscale structures that carry specific cargos and play multiple roles in microbe-host interactions. An appropriate MV isolation method that mimics complex pathogen infections in vivo is needed. After bacterial MVs extraction, flagella or pili can be frequently observed along with MVs by transmission electron microscope (TEM). Recently, MVs from Pseudomonas aeruginosa were found to coexist with Pf4 phages, and this MV-phages complex exhibited a different impact on host cell innate immunity compared with MVs or phages solely. The presence of this MVs-phages complex simulates the real condition of complex pathogen infections within the host. This protocol outlines the extraction of the MVs and Pf4 phages complex of P. aeruginosa PAO1, including the respective isolation and qualification approaches. Our step-by-step bacterial MVs-phages complex extraction protocol provides valuable insights for further studying microbe-host cell interactions and the development of novel phage therapies. Key features • Detailed density gradient extraction procedures of MVs-phages complex • TEM, plaque assay, and PCR to verify the coexistence of MVs and phages • The obtained MVs-phages complex can be used for exploring phage-microbe-host cell interactions Graphical overview.
Insights
Bacterial membrane vesicles (MVs) and phages can coexist, forming a complex that better simulates infections. This study details a protocol for extracting this complex from Pseudomonas aeruginosa, aiding research into microbe-host interactions and phage therapy.
Area of Science:
- Microbiology
- Bacteriology
- Virology
Background:
- Bacterial membrane vesicles (MVs) are nanoscale structures involved in microbe-host interactions.
- Current isolation methods may not fully represent complex pathogen infections.
- The coexistence of MVs and phages, like Pf4 phages with Pseudomonas aeruginosa MVs, is increasingly recognized.
Purpose of the Study:
- To outline a protocol for extracting the MVs-phages complex from Pseudomonas aeruginosa PAO1.
- To provide a method that mimics in vivo conditions of complex pathogen infections.
- To facilitate further research into microbe-host interactions and phage therapy.
Main Methods:
- Density gradient extraction for isolating the MVs-phages complex.
- Transmission electron microscopy (TEM) for visualizing the complex.
- Plaque assays and PCR for verifying the presence and coexistence of MVs and phages.
Main Results:
- A detailed protocol for the extraction of the MVs-phages complex was successfully established.
- Coexistence of MVs and Pf4 phages in the extracted complex was confirmed.
- The extracted complex provides a more biologically relevant model for studying bacterial infections.
Conclusions:
- The developed protocol enables the isolation of a bacterial MVs-phages complex.
- This complex offers a valuable tool for investigating microbe-host cell interactions.
- The findings support the development of novel phage-based therapeutic strategies.
