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Updated: May 23, 2025
Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Published on: April 2, 2013
Visualizing mitochondrial electron transport chain complexes and super-complexes during infection of human
Mariatou Dramé1, Daniel Schator2, Carmen Buchrieser1
1Institut Pasteur, Université Paris Cité, Biologie des Bactéries Intracellulaires and CNRS UMR 6047, Paris, France.
Abstract:
The ultrastructure of mitochondria is pivotal for their respiratory activity. Thus, the regulation of the assembly of the super-complexes (SCs) of the mitochondrial electron transport chain (ETC) might be a core aspect of macrophage immunometabolism during bacterial infection. In order to study the impact of infection by Legionella pneumophila on the configuration of mitochondrial complexes and SCs in human macrophages, we have adapted and combined different methods such as cell sorting of infected cells, magnetic isolation of highly pure and functional mitochondria, quality control of mitochondrial purity by flow cytometry, and BN-PAGE (Blue-Native Polyacrylamide Gel Electrophoresis) coupled to Western Blot using near-infrared (NIR) fluorescence. The here presented protocol uses infected and non-infected human macrophage-like THP-1 cells and GFP-expressing L. pneumophila, but the method can be used to analyze the configuration of ETC complexes and SCs also in other mammalian cells and infected with different intracellular bacteria expressing a fluorescent protein.
Insights
Bacterial infection alters mitochondrial super-complexes in macrophages. This study presents a method to analyze these changes, crucial for understanding macrophage immunometabolism during infection.
Area of Science:
- Immunometabolism
- Mitochondrial biology
- Cellular microbiology
Background:
- Mitochondrial ultrastructure is vital for cellular respiration.
- Super-complexes (SCs) of the mitochondrial electron transport chain (ETC) are key regulators of cellular energy.
- Macrophage immunometabolism is significantly impacted by bacterial infections.
Purpose of the Study:
- To investigate the impact of Legionella pneumophila infection on mitochondrial SC configuration in human macrophages.
- To establish a robust protocol for analyzing ETC SCs in infected immune cells.
Main Methods:
- Combined cell sorting, magnetic isolation of mitochondria, and flow cytometry for purity assessment.
- Utilized Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE) coupled with near-infrared (NIR) fluorescence Western Blot.
- Employed human macrophage-like THP-1 cells and GFP-expressing L. pneumophila.
Main Results:
- The study successfully adapted and combined multiple techniques to analyze mitochondrial SCs in infected macrophages.
- The protocol allows for the detailed examination of ETC complex and SC configuration post-infection.
Conclusions:
- The regulation of mitochondrial SC assembly is a critical component of macrophage immunometabolism during bacterial infections.
- The presented methodology is adaptable for studying various mammalian cells and intracellular bacterial infections involving fluorescent reporters.
