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. 2009 Aug;50(8):3794-801.
doi: 10.1167/iovs.08-3107. Epub 2009 Feb 28.

Properties of PASP: a Pseudomonas protease capable of mediating corneal erosions

Aihua Tang  1 , Mary E Marquart, Jonathan D Fratkin, Clare C McCormick, Armando R Caballero, Hattie P Gatlin, Richard J O'Callaghan
Affiliations

Properties of PASP: a Pseudomonas protease capable of mediating corneal erosions

Aihua Tang et al. Invest Ophthalmol Vis Sci. 2009 Aug.

Abstract

Purpose: To analyze PASP in terms of its gene distribution and expression, its corneal pathologic effects, its enzymatic properties, and the protectiveness of the immune response to this protease.

Methods: Twenty-five strains of P. aeruginosa were analyzed for the PASP gene and secreted protein by PCR and Western blot analysis, respectively. Active recombinant (r)PASP (10 microg/20 microL) or heat-inactivated rPASP was intrastromally injected into rabbit corneas. Pathologic changes were monitored by slit lamp examination (SLE) and histopathology. Purified rPASP was assayed for cleavage of collagens and susceptibility to TLCK. Rabbit antibody to rPASP was produced and tested for enzyme inactivation, and actively immunized rabbits were challenged by intrastromal injection of active rPASP (5 microg).

Results: All 25 strains of P. aeruginosa analyzed were positive for the PASP gene and protein. SLE scores of eyes injected with active rPASP were significantly higher than control eyes at all postinjection times (PI; P <or= 0.004). Histopathologic studies documented the destruction of the corneal epithelial layer and portions of the corneal stroma at 9 hours PI, and polymorphonuclear (PMN) leukocyte infiltration into the cornea by 24 hours after active rPASP injection. PASP cleaved type I and IV collagens and was susceptible to TLCK inhibition. PASP was present in the cytoplasm and periplasm, but only secreted PASP was enzymatically active. A high antibody titer (ELISA titer >or= 10,000) was produced, but this antibody did not protect against active rPASP challenge.

Conclusions: PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.

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Figures

Figure 1
Figure 1
PCR analysis of the PASP gene among multiple P. aeruginosa isolates. Two sets of primers were used: one to amplify the full-length PASP gene (573 bp, A) and the other to amplify a portion of the gene (173 bp, B). Pseudomonas genomic DNA (0.5–1.0 μg) was used as the template in PCR reactions. Lanes 1 and 15: DNA standard (100-bp ladder); lane 2: negative control; lanes 3 to 14: PAO1, PA103, PA103-29, ATCC 19660, PA23, PA24, PA70, PA178, PA13003, PA13004, PA13010, and PA13014, respectively; lanes 16 to 28: PA13036, PA30009, PA30069, PA30099, PA30132, PA30244, PA51003, PA51005, PA51036, PA51037, PA51131, PAALC2, and PAALC3, respectively.
Figure 2
Figure 2
Production and reactivity of antibody to PASP. (A) Antibody titers in immunized rabbits measured by ELISA. Anti-sera were collected after three injections of heat-denatured rPASP. Antibody titers were determined as the midpoint of the titration curve and expressed as the average ± SEM. (B) Western blot analysis of Pseudomonas culture supernatants for the presence of PASP protein. Concentrated P. aeruginosa culture supernatants (30 μL, 2 mg/mL) were electrophoresed and then probed with rabbit polyclonal anti-PASP antibody (1:1000). Lanes 1, 11 and 21: prestained molecular mass standard; lane 2: rPASP; lanes 3 to 10: PAO1, PA103, PA103-29, ATCC 19660, PA23, PA24, PA70, and PA178, respectively; lanes 12 to 20: PA13004, PA13010, PA13014, PA30009, PA30069, PA30099, PA30132, PA30244, and PA51003, respectively; lanes 22 to 29: PA51005, PA51036, PA51037, PA51131, PA13036, PAALC 2, PAALC 3, and PA13003, respectively; lane 30: negative control (E. coli M15 supernatant).
Figure 3
Figure 3
Localization of active PASP. (A) Western blot analysis of the cell lysate, periplasm and culture supernatant of PA103-29 for the presence of PASP. Samples, each containing approximately 60 μg of total protein, were electrophoresed on a 15% SDS-polyacrylamide gel. Lane 1: molecular mass standard; lane 2: rPASP; lane 3: cell lysate; lane 4: periplasm; and lane 5: supernatant. (B) Gelatin zymography for the presence of active PASP in PA103-29. An equal amount of samples (60 μg each) was electrophoresed on a 10% SDS-polyacrylamide gel containing 0.1% gelatin. Lane 1: cell lysate; lane 2: periplasm; and lane 3: supernatant. Only the supernatant sample (lane 3) showed PASP enzyme activity.
Figure 4
Figure 4
Effects of PASP in rabbit eyes. (A) Appearance of eyes. Ten micrograms of active (I) or heat-inactivated (II) rPASP was intrastromally injected into rabbit corneas (six eyes per group). Pathologic changes were monitored at 5, 9, and 24 hours PI. Corneal erosions were stained with fluorescein and photographed under blue light (I). Shown are two representative eyes from the two groups. (B) SLE of PASP-injected rabbit eyes. The eyes were scored by SLE at 5, 9, and 24 hours PI. The mean score ± SEM per group was calculated. SLE scores of active rPASP injected eyes were significantly higher than the ones injected with heat-inactivated enzyme at any time point (P ≤ 0.004).
Figure 5
Figure 5
Histopathologic analysis of PASP effects in rabbit eyes. Ten micrograms of active (A–C) or heat-inactivated (D) rPASP was intrastromally injected into rabbit corneas. Eyes were harvested at 9 (A, B) and 24 (C, D) hours after injection. At 9 hours PI, eyes with active rPASP showed corneal erosions (A, B) and an erosion extending into the stroma (B). Intense corneal infiltrate in the eye with active rPASP was present at 24 hours PI (C). Eyes with heat-inactivated rPASP showed normal corneal structure (D). Magnifications: (A–C) ×40 and ×200; (D) ×40 and ×400.
Figure 6
Figure 6
Fluorescence assays of gelatin and collagen digestion by P. aeruginosa proteases. Gelatin, type I or type IV collagen was incubated with either 6 μg of rPASP or 0.1 μg of LasB, AP, LasA, and rPIV individually. Fluorescence was measured at 1, 3, 5, and 24 hours after incubation. Background fluorescence, determined by no-enzyme reactions, was subtracted from each value. Data are expressed as the mean ± SEM. Experiments were performed in triplicate and repeated once.
Figure 7
Figure 7
In vitro and in vivo reactions of PASP with anti-PASP antibody. (A) In vitro casein fluorescence assay. Active rPASP (10 μg in 20 μL) was incubated with anti-PASP IgG (20 μL; 1.3 mg/mL) antibody overnight. Fluorescent casein was then added to test the enzymatic activity. Fluorescence was measured at various time points. IgG from mock anti-sera (20 μL; 1.2 mg/mL) served as the control. Data are expressed as the mean ± SEM. Experiments were performed in triplicate and repeated once. (B) SLE scores of rPASP-challenged rabbit eyes. Active rPASP (5 μg in 10 μL) was used to challenge the rabbits by intrastromal injection. Mock-immunized rabbits (n = 3) did not produce significantly higher SLE scores in the eye than did rPASP-immunized rabbits (n = 3) at either 7 or 24 hours PI (P ≥ 0.05). Data are expressed as the mean SLE score ± SEM.
Figure 8
Figure 8
Inhibition of PASP activity by TLCK. Fluorescein-conjugated gelatin was incubated with rPASP (10 μg) alone or with rPASP plus various concentrations of TLCK. Fluorescence was measured after 24 hours. Assays were performed in triplicate. Data are expressed as mean fluorescence value ± SEM.
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