Related Experiment Videos
Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement
M B Goldberg1, O Bârzu, C Parsot
1Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.
Insights
Shigella flexneri protein IcsA binds and hydrolyzes ATP, localizing to the host cell actin tail. This protein is secreted in a cleaved form, aiding bacterial cell-to-cell spread.
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Pathogenesis
Background:
- Shigella flexneri invades host cells and utilizes the host cytoskeleton for motility.
- The bacterial protein IcsA is crucial for mediating this intracellular and intercellular movement.
Purpose of the Study:
- To investigate the biochemical properties and localization of Shigella flexneri's IcsA protein.
- To elucidate the mechanism by which IcsA interacts with the host cell cytoskeleton.
Main Methods:
- Purification of IcsA protein for in vitro studies.
- ATP binding and hydrolysis assays.
- Immunofluorescence labeling of infected HeLa cells and bacterial surface localization studies.
- Analysis of IcsA secretion in vitro.
Main Results:
- Purified IcsA binds and hydrolyzes ATP.
- IcsA exhibits unipolar surface localization on S. flexneri.
- IcsA localizes to the actin tails in infected HeLa cells.
- IcsA is secreted in a cleaved form into the culture supernatant.
Conclusions:
- IcsA possesses ATPase activity, suggesting a role in energy-dependent processes.
- The unipolar localization and association with actin tails indicate IcsA's direct involvement in host cell actin-based motility.
- Secretion of cleaved IcsA supports its function in facilitating bacterial spread within host tissues.
Abstract:
Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.