4.6 Article

Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

期刊

FRONTIERS IN MICROBIOLOGY
卷 12, 期 -, 页码 -

出版社

FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2021.654193

关键词

Helicobacter pylori; bacterial outer membrane vesicles; defined growth medium; OMVs isolation method; proteomics

资金

  1. Norte Portugal Regional Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) [NORTE-01-0145-FEDER000029]
  2. Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance [ROTEIRO/0028/2013, LISBOA-01-0145-FEDER-022125]
  3. FCT - Fundacao para a Ciencia e a Tecnologia [SFRH/BD/116965/2016, SFRH/BDP/110065/2015]
  4. FCT RJEC [3762]
  5. [PPBIPOCI-01-0145-FEDER-022122]

向作者/读者索取更多资源

The study presents a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium, yielding highly pure OMVs from cultures of different H. pylori strains and growth periods. Analysis of the proteome of OMVs revealed differentially expressed proteins, including VacA, suggesting the reliability of the protocol.
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 mu m 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.

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