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Method for the Isolation of Francisella tularensis Outer Membranes
Published on: June 29, 2010
The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella
Audrey Chong1, Tara D Wehrly, Vinod Nair
1National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, 903 South 4th Street, Hamilton, MT 59840, USA.
Abstract:
Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.
Insights
Francisella tularensis escapes macrophages by disrupting phagosomes. Early phagosome maturation, including acidification, is crucial for optimal escape and intracellular growth of this pathogen.
Area of Science:
- Microbiology
- Cell Biology
- Pathogen-Host Interactions
Background:
- Francisella tularensis is an intracellular bacterium that replicates within host macrophages.
- Efficient intracellular survival requires F. tularensis to escape its initial phagosome into the cytoplasm.
Purpose of the Study:
- To investigate the role of early phagosome maturation in the intracellular lifecycle of F. tularensis.
- To characterize the early trafficking events of F. tularensis subsp. tularensis strain Schu S4 within macrophages.
Main Methods:
- Utilized a murine bone marrow-derived macrophage model.
- Inhibited endosomal acidification using vacuolar ATPase inhibitors (bafilomycin A1, concanamycin A).
- Analyzed phagosomal escape, cytosolic replication, and Francisella pathogenicity island (FPI) protein expression.
Main Results:
- Early phagosomes containing Schu S4 transiently interacted with endosomes and acidified before disruption.
- Inhibition of endosomal acidification delayed but did not prevent phagosomal escape and replication.
- FPI protein expression was induced independently of phagosomal pH, suggesting alternative signaling cues.
Conclusions:
- Early phagosomal maturation, including acidification, is essential for optimal phagosomal escape and intracellular growth of F. tularensis.
- The early phagosome provides non-pH-dependent signals that regulate the intracellular induction of FPI proteins, critical for pathogenesis.
