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Purification of Pathogen Vacuoles from Legionella-infected Phagocytes
09:25

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Published on: June 19, 2012

Isolation of pathogen-containing vacuoles

Olga Shevchuk1, Michael Steinert

  • 1Institut für Mikrobiologie, Technische Universität Braunschweig, Braunschweig, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2013
PubMed

Insights

This study introduces a new method to isolate pathogen-containing vacuoles (PCVs) from Dictyostelium discoideum. This technique aids in understanding how bacteria like Legionella pneumophila survive inside host cells.

Area of Science:

  • Cell Biology
  • Microbiology
  • Biochemistry

Background:

  • Dictyostelium discoideum are professional phagocytes, engulfing various particles.
  • Bacterial pathogens can survive within D. discoideum vacuoles by manipulating host pathways.

Purpose of the Study:

  • To develop a method for isolating pathogen-containing vacuoles (PCVs).
  • To investigate molecular mechanisms of intracellular pathogen survival.

Main Methods:

  • Infection of D. discoideum with Legionella pneumophila.
  • Isolation of PCVs using colloidal iron loading, mechanical lysis, INT labeling, Benzonase treatment, centrifugation, and magnetic separation.
  • Subcellular fractionation via OptiPrep density gradients.

Main Results:

  • High-purity PCVs were successfully isolated.
  • Proteins from PCVs were analyzed by mass spectrometry, revealing pathogen-directed vacuole maturation.
  • The method allows for subsequent protein modification and lipidome analyses.

Conclusions:

  • The developed method enables the isolation and analysis of PCVs.
  • This facilitates the study of host-pathogen interactions and vacuole dynamics.
  • The technique is versatile for further proteomic and lipidomic investigations.

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