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Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria
Published on: October 31, 2019
Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically
Joana Melo1,2,3, Vanessa Pinto1,2, Tânia Fernandes1,2
1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
Abstract:
Outer membrane vesicles (OMVs) are small vesicles constitutively shed by all Gram-negative bacterium, which have been proposed to play a role in Helicobacter pylori persistence and pathogenesis. The methods currently available for the isolation of H. pylori OMVs are diverse and time-consuming, raising the need for a protocol standardization, which was the main aim of this study. Here, we showed that the chemically defined F12 medium, supplemented with cholesterol, nutritionally supports bacterial growth and maintains H. pylori viability for at least 72 h. Additionally, we developed an abridged protocol for isolation of OMVs from these bacterial cultures, which comprises a low-speed centrifugation, supernatant filtration through a 0.45 μm pore, and two ultracentrifugations for OMVs' recovery and washing. Using this approach, a good yield of highly pure bona fide OMVs was recovered from cultures of different H. pylori strains and in different periods of bacterial growth, as assessed by nanoparticle tracking analysis, transmission electron microscopy (TEM), and proteomic analyses, confirming the reliability of the protocol. Analysis of the proteome of OMVs isolated from H. pylori F12-cholesterol cultures at different time points of bacterial growth revealed differentially expressed proteins, including the vacuolating cytotoxin VacA. In conclusion, this work proposes a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium that is suitable for implementation in research and in the biopharmaceutical field.
Insights
This study presents a standardized, cost-effective protocol for isolating outer membrane vesicles (OMVs) from Helicobacter pylori. The new method ensures high purity and yield, crucial for H. pylori research and biopharmaceutical applications.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Outer membrane vesicles (OMVs) are implicated in Helicobacter pylori pathogenesis and persistence.
- Current OMV isolation methods for H. pylori are inefficient and lack standardization.
Purpose of the Study:
- To develop and validate a standardized, time- and cost-efficient protocol for H. pylori OMV isolation.
- To assess the purity and yield of OMVs obtained using the novel protocol.
Main Methods:
- Culturing H. pylori in chemically defined F12 medium with cholesterol.
- Employing a streamlined isolation protocol: low-speed centrifugation, 0.45 μm filtration, and dual ultracentrifugation.
- Characterizing isolated OMVs using nanoparticle tracking analysis, TEM, and proteomic analysis.
Main Results:
- The F12 medium with cholesterol supports H. pylori growth and viability for over 72 hours.
- The abridged protocol yields pure H. pylori OMVs from various strains and growth phases.
- Proteomic analysis identified differentially expressed proteins, including VacA, in isolated OMVs.
Conclusions:
- A reliable, efficient, and cost-effective protocol for H. pylori OMV isolation is established.
- The protocol is suitable for research and biopharmaceutical applications.
- The method facilitates further investigation into H. pylori OMV roles in pathogenesis.
