Cleavage and activation of corneal matrix metalloproteases by Pseudomonas aeruginosa proteases.
OBJECTIVE
To examine the effect of Pseudomonas aeruginosa on the expression of corneal matrix metalloproteases and the effect of its proteases on activation of corneal matrix metalloproteases in vitro.


METHODS
Rat corneas and human corneal fibroblasts were co-cultivated with two different strains (RPS & 599A) of P. aeruginosa and one strain of Staphylococcus aureus, and the conditioned media were analyzed for proteolytic activity by gelatin and casein zymography. Human corneal fibroblast-conditioned medium was incubated with that from either strain of P. aeruginosa and was analyzed in a similar manner.


RESULTS
Normal rat corneas in organ culture produce a 65 kDa gelatinase (inactive matrix metalloprotease-2), whereas thermally injured rat corneas additionally produce gelatinases with molecular masses of 92 kDa (inactive matrix metalloproteases-9) and > 200 kDa. Matrix metalloprotease-2 is also detected in human corneal fibroblast-conditioned medium. Although these matrix metalloproteases are no longer detectable when rat corneas or human corneal fibroblasts are co-cultured with two strains of P. aeruginosa for 48 hr, a 58 kDa gelatinase fragment appears in earlier stages of co-culture. In contrast, S. aureus does not affect matrix metalloprotease-2. The 58 kDa fragment is also evident by incubating human corneal fibroblast-conditioned medium with that from either strain of P. aeruginosa. Conditioned medium from the RPS strain, which produces both elastase and alkaline protease, is more effective in cleaving matrix metalloprotease-2 than that from the 599A strain, which expresses mainly alkaline protease.


CONCLUSIONS
The secreted inactive corneal matrix metalloprotease-2 is activated through limited proteolysis by pseudomonal proteases.
Matsumoto
K
K
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Shams
N B
NB
Hanninen
L A
LA
Kenyon
K R
KR
D000818:Animals; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D003315:Cornea / Q000201:enzymology* / Q000382:microbiology; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology / Q000382:microbiology; D006801:Humans; D008297:Male; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000378:metabolism*; D009924:Organ Culture Techniques; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000494:pharmacology*
D003470:Culture Media; C038495:Pseudomonas serine proteinase; D012697:Serine Endopeptidases; D017364:Collagenases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9