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Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro
S M Fleiszig1, T S Zaidi, G B Pier
1Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Abstract:
Pseudomonas aeruginosa is usually considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have previously demonstrated that P. aeruginosa is able to invade corneal cells during infectious keratitis in mice. In vitro, P. aeruginosa was found to enter the following cells: human corneal cells removed by irrigation; epithelial cells in the cornea of rats, mice, and rabbits; and primary corneal epithelial cells cultured from rat and rabbit eyes. The level of invasion was related to the level of adherent or associated bacteria. In general, invasion was more efficient with cultured epithelial cells than with cells tested in situ. Invasion did not occur when assays were performed at 4 degrees C. Cytochalasin D but not colchicine inhibited bacterial invasion, suggesting that bacterial entry was an endocytic process dependent on actin microfilaments but not microtubules. Bacteria that invaded cultured corneal epithelial cells were found to multiply within cells. The ability of P. aeruginosa to invade and multiply within corneal epithelial cells may contribute to the virulence of this organism during infectious keratitis, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.
Insights
Pseudomonas aeruginosa can invade and multiply within corneal epithelial cells, challenging its role as solely an extracellular pathogen. This intracellular behavior may enhance virulence in infectious keratitis.
Area of Science:
- Microbiology
- Ophthalmology
- Cell Biology
Background:
- Pseudomonas aeruginosa is typically viewed as an extracellular bacterial pathogen.
- Previous studies suggested P. aeruginosa invades corneal cells during infectious keratitis in mice.
Purpose of the Study:
- To investigate the capacity of Pseudomonas aeruginosa to invade and survive within corneal epithelial cells.
- To elucidate the mechanism and implications of P. aeruginosa corneal cell invasion.
Main Methods:
- In vitro and in vivo assays using gentamicin to assess intracellular bacterial survival.
- Invasion assays performed on various corneal cell types (human, rat, mouse, rabbit) in situ and in culture.
- Treatment with cytochalasin D and colchicine to determine the mechanism of bacterial entry.
Main Results:
- P. aeruginosa demonstrated invasion into human, rat, mouse, and rabbit corneal epithelial cells.
- Bacterial invasion efficiency correlated with bacterial adherence.
- Invasion was an active, endocytic process dependent on actin microfilaments, inhibited at 4°C.
- Invaded bacteria multiplied within cultured corneal epithelial cells.
Conclusions:
- Pseudomonas aeruginosa possesses the ability to invade and replicate within corneal epithelial cells.
- This intracellular capability may contribute to P. aeruginosa virulence in infectious keratitis.
- Intracellular bacteria can evade host defenses and antibiotic treatments.