Properties of PASP: a Pseudomonas protease capable of mediating corneal erosions.
OBJECTIVE
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.
Tang
Aihua
A
Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Marquart
Mary E
ME
Fratkin
Jonathan D
JD
McCormick
Clare C
CC
Caballero
Armando R
AR
Gatlin
Hattie P
HP
O'Callaghan
Richard J
RJ
D000818:Animals; D015153:Blotting, Western; D002634:Chemotaxis, Leukocyte; D003094:Collagen / Q000378:metabolism; D003315:Cornea / Q000187:drug effects / Q000378:metabolism; D003316:Corneal Diseases / Q000201:enzymology / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay; D015818:Eye Infections, Bacterial / Q000201:enzymology / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D007267:Injections; D009504:Neutrophils / Q000502:physiology; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000201:enzymology / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000502:physiology*
D011994:Recombinant Proteins; D003094:Collagen; C038495:Pseudomonas serine proteinase; D012697:Serine Endopeptidases