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Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa
Z Dong1, M Katar, B E Linebaugh
1Department of Immunology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Abstract:
C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.
Insights
Pseudomonas aeruginosa infection significantly increases cathepsin B, D, and L activity in mouse corneas, contributing to tissue damage. Immunization reduces this inflammatory response and cathepsin upregulation.
Area of Science:
- Ophthalmology
- Microbiology
- Biochemistry
Background:
- Pseudomonas aeruginosa is a common cause of bacterial keratitis.
- Cathepsins are proteases involved in tissue remodeling and inflammation.
- Understanding the role of cathepsins in bacterial keratitis is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the role of cathepsins B, D, and L in Pseudomonas aeruginosa keratitis.
- To determine the effect of immunization on cathepsin expression and activity during infection.
- To elucidate the mechanism of corneal destruction mediated by cathepsins.
Main Methods:
- Semi-quantitative RT-PCR to detect cathepsin gene expression.
- Immunoblot analysis to confirm protein expression levels.
- Peptidase assays to measure cathepsin enzymatic activities.
- Immunohistochemical staining to localize cathepsin expression in corneal tissues.
Main Results:
- Cathepsins B, D, and L were detected in normal corneas with basal enzymatic activity.
- P. aeruginosa infection in naïve mice significantly upregulated cathepsin B, D, and L gene and protein expression and enzymatic activity, peaking around day 6 postinfection.
- Immunization against P. aeruginosa attenuated the induction of cathepsin expression and activity.
- Induced cathepsin expression shifted from the epithelium to the substantia propria in infected corneas.
Conclusions:
- Up-regulated cathepsin B, D, and L activity correlates with the inflammatory response in P. aeruginosa keratitis.
- Immunization effectively reduces the P. aeruginosa-induced cathepsin upregulation.
- Cathepsin-mediated extracellular matrix degradation is a likely mechanism for corneal destruction during P. aeruginosa infection.
