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

Vanessa Pinto,Hugo Osório,Tânia Fernandes,Joana Melo,Ceu Figueiredo,Ana R Malheiro,Marina Leite

doi:10.3389/fmicb.2021.654193

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.

Highlights

Helicobacter pylori infects about half of the human population and remains the most common chronic infection in the world (Hooi et al, 2017)
The bacterial growth of H. pylori 26695 was monitored by measuring the optical density at 600 nm (OD600) of the bacterial liquid cultures at 24, 48, 64, and 72 h, starting with an inoculum density of 0.02 per mL at OD600 (∼6 × 106 CFUs/mL) in 200 mL of F12-cholesterol medium (Figure 1A)
The growth kinetics of H. pylori 26695 in the chemically defined F12-cholesterol medium and in the complex Brucella Broth (BB) medium supplemented with 5% fetal bovine serum (FBS) was compared, under the same experimental conditions (Supplementary Figure 1A)

Summary

Introduction

Helicobacter pylori infects about half of the human population and remains the most common chronic infection in the world (Hooi et al, 2017). This Gram-negative bacterium is acquired in childhood, and unless treated, it establishes a life-long infection of the host gastric mucosa. Isolation and Characterization of H. pylori OMVs disease and gastric cancer, both adenocarcinoma and mucosaassociated lymphoid tissue (MALT) lymphoma (Atherton, 2006; Cover and Blaser, 2009). H. pylori-mediated diseases, as well as bacterial colonization, survival, and persistence in the gastric mucosa depend upon the concerted action of multiple virulence factors and mechanisms (Blaser and Atherton, 2004). The full contribution of OMVs to bacteria-host and bacteria-bacteria interactions is yet to be disclosed, considering that their effects may extend to locations distant from the locally circumscribed bacterial niche (Kulp and Kuehn, 2010)

Methods

Results

Conclusion