Related Experiment Videos
Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells
Manuel R Amieva1, Nina R Salama, Lucy S Tompkins
1Department of Microbiology and Immunology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305, USA. amieva@stanford.edu
Abstract:
Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori-containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA- mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.
Insights
Helicobacter pylori can survive inside epithelial cells by entering large vacuoles, remaining viable and motile. This intracellular survival in a vacuolar niche may explain why H. pylori infections are difficult to eradicate.
Area of Science:
- Microbiology
- Cell Biology
- Infectious Diseases
Background:
- Intracellular Helicobacter pylori have been observed, but their fate and survival mechanisms remain unclear.
- Understanding H. pylori's intracellular behavior is crucial for developing effective eradication strategies.
Purpose of the Study:
- To investigate the fate and survival of intracellular Helicobacter pylori within host epithelial cells.
- To elucidate the cellular mechanisms and niche exploited by H. pylori for intracellular survival.
Main Methods:
- Utilized differential interference contrast (DIC) video and immunofluorescence microscopy to observe H. pylori within cells.
- Employed time-lapse microscopy and pulsed gentamicin treatments to track bacterial viability and survival over time.
- Used electron microscopy and endosomal dye staining to characterize the morphology of H. pylori-containing vacuoles.
Main Results:
- A proportion of cell-associated H. pylori enter large cytoplasmic vacuoles in various epithelial cell lines.
- Intravacuolar H. pylori remain viable, motile, and protected from extracellular gentamicin, with a half-life of approximately 24 hours.
- These vacuoles resemble late endosomal multivesicular bodies, and bacterial entry/survival is independent of VacA.
Conclusions:
- Helicobacter pylori exploits an intravacuolar niche within host epithelial cells for survival.
- This intracellular survival mechanism, involving vacuole formation and protection, contributes to the persistence and difficulty in eradicating H. pylori infections.