Further Analysis of Pseudomonas aeruginosa Small Protease (PASP): a Corneal Virulence Factor

Abstract

Purpose: : To further characterize the newly discovered Pseudomonas aeruginosa small protease (PASP) as a corneal virulence factor, efforts were made to improve the in vitro assay of the enzyme, to analyze its histopathological effects on rabbit corneas, and determine the extent and protectiveness of the immune response to the protease.

Methods: : Chromogenic or fluorescent substrates were incubated with 10 ug active recombinant PASP (rPASP), heat-inactivated rPASP, or an equal volume of dH₂O at 37 °C overnight. Optical density (OD) at 410 nm or fluorescence at 515 nm was measured after incubation. Active rPASP (10 ug /20 ul) or an equal amount of heat-inactivated rPASP was intrastromally injected into rabbit corneas and pathological changes were monitored by slit lamp examination (SLE) at various time points post-injection (PI). Histopathological analysis was also performed on rabbit eyes after enzyme injection. Purified rPASP was used to immunize rabbits and antibody titers were determined by ELISA. Both rPASP- and mock-immunized rabbits were challenged with 5 ug active rPASP by intrastromal injection and pathological changes were analyzed.

Results: : More than thirty chromogenic peptide substrates were screened for their susceptibility to PASP digestion and only Gly-Arg-p-nitroanilide was found to be digested. Fluorescein conjugated casein, collagen, and gelatin were also susceptible to rPASP digestion. Intrastromal injection of rPASP caused corneal epithelial erosions and severe inflammation. The SLE scores of eyes injected with active rPASP were significantly higher than the control eyes at 5, 9, and 24 hours PI examined (P ≤ 0.004). Histopathological studies documented polymorphonuclear (PMN) leukocyte infiltration into the cornea and also the anterior chamber and limbus. Immunization with rPASP produced a very high titer of antibody (4.1 ± 0.1, log₁₀ value ± SEM), but, the rPASP-immunized rabbits were not protected from pathological changes following injection of active rPASP.

Conclusions: : PASP studies have been facilitated by assays. Recombinant PASP caused corneal epithelial erosions and an intense inflammatory response. Hosts with high titer antibody were not protected from extensive pathologic changes following injection of active rPASP.

Keywords: keratitis • pseudomonas • microbial pathogenesis: experimental studies