Live cell imaging of phosphoinositide dynamics during Legionella infection
1Max von Pettenkofer Institute, Ludwig-Maximilians University, Pettenkoferstraße 9a, 80336, Munich, Germany.
Abstract:
The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.
Insights
Legionella pneumophila manipulates host cell vacuoles using effector proteins. This study details a method using amoeba models to visualize and study these bacterial-induced membrane changes in real-time during infection.
Area of Science:
- Microbiology
- Cell Biology
- Molecular Biology
Background:
- Legionella pneumophila is an intracellular pathogen that replicates within a specialized vacuole.
- The Icm/Dot type IV secretion system translocates numerous effector proteins into host cells.
- Bacterial effector proteins bind to phosphoinositide (PI) lipids on the vacuole membrane, controlling localization and host factor recruitment.
Purpose of the Study:
- To describe a protocol for observing phosphoinositide patterns on the Legionella-containing vacuole (LCV) membrane.
- To utilize amoeba model systems for live cell imaging of LCV dynamics.
- To study the real-time changes in membrane PI patterns during Legionella infection.
Main Methods:
- Construction and handling of amoeba (Dictyostelium discoideum) model systems expressing fluorescent protein probes.
- Live cell imaging techniques to capture rapid changes in membrane PI patterning.
- Observation of LCV PI dynamics throughout the course of Legionella pneumophila infection.
Main Results:
- Demonstration of a protocol to visualize host-pathogen interactions at the vacuolar membrane.
- Real-time tracking of phosphoinositide dynamics on the LCV.
- Insights into how Legionella pneumophila modulates host vacuole membranes.
Conclusions:
- Amoeba-based live cell imaging provides a powerful tool for studying pathogen-host interactions.
- Understanding LCV phosphoinositide dynamics is crucial for deciphering Legionella pathogenesis.
- The described protocol facilitates real-time observation of bacterial effector function and host cell manipulation.
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