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Watson DC, Johnson S, Santos A, et al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J Vis Exp. 2021;(176)doi: 10.3791/63155.
Watson, D. C., Johnson, S., Santos, A., Yin, M., Bayik, D., Lathia, J. D., & Dwidar, M. (2021). Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. Journal of visualized experiments : JoVE, (176), . https://doi.org/10.3791/63155
Watson, Dionysios C, et al. "Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria." Journal of visualized experiments : JoVE vol. ,176 (2021). doi: https://doi.org/10.3791/63155
Watson DC, Johnson S, Santos A, Yin M, Bayik D, Lathia JD, Dwidar M. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J Vis Exp. 2021 Oct 13;(176). doi: 10.3791/63155. PMID: 34723953; PMCID: PMC8729794.
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J Vis Exp. 2021 Oct 13;(176). doi: 10.3791/63155.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria.

Journal of visualized experiments : JoVE

Dionysios C Watson, Sadie Johnson, Akeem Santos, Mei Yin, Defne Bayik, Justin D Lathia, Mohammed Dwidar

Affiliations

  1. Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic; University Hospitals Cleveland Medical Center; Case Western Reserve University; [email protected].
  2. Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic.
  3. Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic; Center for Microbiome & Human Health, Cleveland Clinic.
  4. Electron Microscopy Core, Lerner Research Institute, Cleveland Clinic.
  5. Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic; Case Western Reserve University.
  6. Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic; Case Western Reserve University; Center for Microbiome & Human Health, Cleveland Clinic; [email protected].

PMID: 34723953 PMCID: PMC8729794 DOI: 10.3791/63155

Abstract

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

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