Related Experiment Videos
Pasteurella multocida enters polarized epithelial cells by interacting with host F-actin
M J Rabier1, N K Tyler, N J Walker
1Department of Anatomy, School of Veterinary Medicine, University of California at Davis 95616, USA.
Abstract:
We investigated the interaction of an avian strain of Pasteurella multocida with the cytoskeleton of MDCK cells, which formed a polarized epithelium when grown on type I collagen coated filters. Bacteria were incubated with MDCK cells for 30 min. 2, 4 and 6 hours and their location and association with the cell cytoskeleton determined by double-label immunofluorescence confocal microscopy. Cells were stained with a polyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) actin. Confocal microscopy revealed that bacteria entered the cells by 30 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Electron microscopy demonstrated that by 2 hours, each bacterium was associated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection disrupted the actin cytoskeleton and inhibited entry of P. multocida.
Insights
Avian Pasteurella multocida invades host cells and reorganizes the actin cytoskeleton. Disrupting actin filaments with cytochalasin D inhibits bacterial entry, highlighting the cytoskeleton's role in infection.
Area of Science:
- Microbiology
- Cell Biology
- Pathogen-Host Interactions
Background:
- Polarized epithelial cells, such as Madin-Darby Canine Kidney (MDCK) cells grown on collagen, serve as models for studying host-pathogen interactions.
- The cytoskeleton, particularly filamentous (F) actin, plays a crucial role in cellular processes including pathogen entry.
- Pasteurella multocida is an avian pathogen that can cause significant disease.
Purpose of the Study:
- To investigate the interaction between an avian strain of Pasteurella multocida and the cytoskeleton of MDCK cells.
- To determine the temporal dynamics of bacterial entry and its association with the actin cytoskeleton.
- To elucidate the role of the actin cytoskeleton in Pasteurella multocida infection.
Main Methods:
- MDCK cells grown on collagen-coated filters were infected with Pasteurella multocida for varying durations (30 min to 6 hours).
- Double-label immunofluorescence confocal microscopy was used to visualize bacteria and F-actin distribution.
- Electron microscopy was employed to examine the ultrastructural association between bacteria and cytoskeletal filaments.
- Cytochalasin D was used to disrupt the actin cytoskeleton and assess its effect on bacterial entry.
Main Results:
- Pasteurella multocida entered MDCK cells within 30 minutes of incubation.
- By 6 hours post-infection, the actin cytoskeleton showed significant reorganization, with long filaments replaced by discrete foci containing bacteria.
- Electron microscopy revealed bacteria associated with short 5-6 nm filaments within 2 hours.
- Pre-treatment with cytochalasin D disrupted the actin cytoskeleton and significantly inhibited Pasteurella multocida entry.
Conclusions:
- Avian Pasteurella multocida interacts with and alters the actin cytoskeleton of MDCK cells during infection.
- Bacterial entry is dependent on an intact actin cytoskeleton.
- The findings provide insights into the mechanism of Pasteurella multocida pathogenesis at the cellular level.