The next step in corneal virulence is likely to be destruction of host tissue by the bacterium, and this may be mediated by toxins or proteases. Salicylic acid was capable of reducing the production of extracellular proteases in
P. aeruginosa. The amounts of protease IV and elastase in the culture supernatants of
P. aeruginosa exposed to salicylic acid were reduced compared with the control. Using plate assays for total protease (plates containing skim milk) or for elastase (plates containing gelatin), Prithiviraj et al.
39 have also demonstrated a significant decrease in these activities in the presence of subinhibitory concentrations of salicylic acid for
P. aeruginosa PA14. Several studies have demonstrated that
Pseudomonas serine protease IV is a significant virulence factor during corneal infections.
18 19 46 Further, Caballero et al.
56 suggest that the ability of
P. aeruginosa to destroy elastin is a major virulence determinant during acute infection, and increased protease activities were associated with tissue damage. Therefore, a significant decrease in protease IV and elastase production with salicylic acid suggests that salicylic acid may reduce corneal virulence and the inflammatory response of the eye during corneal infection. The appearance of an unknown enzyme activity just above the alkaline protease seen in the gel may be an uncharacterized protease expressed in response to salicylic acid exposure. The characteristics of this protease band remain to be defined, but we have demonstrated very low levels of a protease of apparent similar molecular mass (98 kDa) in strain 6294 in other experiments.
30 There has been a report of a protease of ∼80 kDa called PASP produced by
P. aeruginosa PA103 and produced from the gene
PA0423. This protease can be seen in a mutant that is deficient in protease IV activity.
57 In other experiments, if biofilms of
P. aeruginosa are exposed to ciprofloxacin, there is a 38% to 65% decrease in total proteolytic activity.
58 In that study
58 P. aeruginosa strain PA1230 was shown to have a protease profile on zymography very similar to the profile of 6294 in the present study. Of note, although there was no report of additional protease bands appearing during treatment, more total protease was reported to be produced in response to incubation of biofilms in two times the MIC.
58 Most of the protease experiments were conducted at a temperature of 37°C in vitro, whereas the temperature of the cornea is approximately 33°C.
59 This difference is unlikely to affect the results; indeed protease IV, for example, of
P. aeruginosa is active in the corneas of mice.
19