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Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro
S Miyajima1, T Akaike, K Matsumoto
1Department of Microbiology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.
Abstract:
The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines. Corneal lesions and MMP induction in vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa. Effects of pseudomonal virulence factors [elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)], tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting. Corneal ulcers with typical ring abscesses were observed 12-24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas. Pseudomonal elastase caused the most extensive damage to both cell types. RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9. Exotoxin A had no effect on MMP expression. Both IL-1beta and TNF-alpha augmented MMP-9 expression in HT1080 cells. Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells. In conclusion, corneal destruction seen with P. aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase.
Insights
Pseudomonas aeruginosa infection causes corneal damage by increasing matrix metalloproteinases (MMPs) through bacterial factors and inflammation. These MMPs are activated by pseudomonal elastase, leading to corneal destruction.
Area of Science:
- Ophthalmology
- Microbiology
- Cell Biology
Background:
- Pseudomonas aeruginosa is a major cause of bacterial keratitis.
- Matrix metalloproteinases (MMPs) play a role in tissue degradation during corneal infections.
Purpose of the Study:
- To investigate the role of Pseudomonas aeruginosa virulence factors and inflammatory cytokines in inducing and activating MMPs in the cornea.
- To elucidate the mechanisms of corneal destruction in pseudomonal keratitis.
Main Methods:
- In vivo studies using rabbit corneas infected with P. aeruginosa.
- In vitro studies using rabbit corneal fibroblasts and human fibrosarcoma cells.
- Techniques included reverse transcriptase-polymerase chain reaction (RT-PCR), zymography, and immunoblotting.
Main Results:
- P. aeruginosa infection induced corneal ulcers and upregulated MMPs, especially MMP-9.
- Pseudomonal elastase caused significant damage and activated MMP-2 and MMP-9.
- Proinflammatory cytokines (TNF-alpha, IL-1beta) augmented MMP-9 expression.
Conclusions:
- Corneal destruction in P. aeruginosa keratitis is mediated by enhanced MMP expression and activation.
- Pseudomonal exoproteases and inflammatory cytokines stimulate corneal cells to produce MMPs.
- Pseudomonal elastase is a key factor in the proteolytic activation of MMPs, contributing to tissue damage.