Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins

Abstract

Purpose: To determine the effects of exoproducts from the corneal pathogen Pseudomonas aeruginosa on corneal proteinases and proteins.

Methods: Whole rabbit corneas were cultured in the presence or absence of broths conditioned with Pseudomonas aeruginosa, elastase, alkaline protease, and exotoxin A. Protein synthesis was assayed by adding 35S-methionine during the last 6 hours of culture. Caseinolytic assays and zymography on sodium dodecyl sulfate polyacrylamide gels containing casein and gelatin were used in the presence and absence of inhibitors to quantify and identify corneal proteinases.

Results: The major proteinases released by the corneas were 92/89 kD (MMP9) and 65 kD (72 kD gelatinase, MMP2) gelatinases and a 97 kD caseinase. Minor proteinases observed included 184, 166, 156, 153, 126, 111, 102, 60, 57, and 43 kD gelatinases and 170, 136, 85, and 54 kD caseinases. P. aeruginosa elastase at 1 microgram/ml cleaved the 92 kD gelatinase to yield a 77 kD active form and cleaved the 65 kD gelatinase to yield a 57 kD active form. At 25 micrograms/ml elastase, the gelatinases were degraded. P. aeruginosa alkaline protease had no effect on the 92 or 65 kD gelatinases. Both elastase and alkaline protease degraded the 97 kD caseinase. Proteinases other than elastase and alkaline protease in P. aeruginosa103- and P. aeruginosa01-conditioned broths also activated and/or degraded corneal proteinases. Exotoxin A inhibited the synthesis of the 92 kD gelatinase and most other proteins. The 72 kD gelatinase and the 97 kD caseinase were released in the presence of exotoxin A.

Conclusions: Pseudomonas aeruginosa exoproducts can contribute directly to keratitis caused by Pseudomonas organisms through toxic effects on corneal cells and degradation of corneal proteins and indirectly through the activation of corneal proteinases.