Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions.
OBJECTIVE
The purpose of this study was to identify a new Pseudomonas protease and determine its possible role in keratitis.


METHODS
Concentrated culture supernatants of the Pseudomonas aeruginosa strains PA103 and ATCC 19660 were analyzed by zymography. P. aeruginosa small protease (PASP) was purified from strain PA103, and modified elastase B (LasB) was purified from strain ATCC 19660. SDS-PAGE and Western blot analysis were performed on purified PASP and modified LasB. PASP was further analyzed by mass spectrometry and amino-terminal sequencing. The Pasp gene was cloned and expressed, affinity-purified in denatured form from inclusion bodies, and refolded by removal of the denaturant. Purified recombinant PASP was analyzed by zymography for protease activity. PASP and heat-inactivated PASP were injected into rabbit corneas, and the corneas were monitored for erosions caused by protease activity.


RESULTS
Each strain produced a protease with a molecular mass of 80 kDa on zymograms. LasB antiserum identified the ATCC 19660 protease as modified LasB. Mass spectrometry defined the PA103 protease as having a molecular mass of 18.5 kDa. Amino-terminal sequencing and analysis of the P. aeruginosa genome sequence determined that the PA103 Pasp gene sequence was >99% identical with the PA0423 sequence of strain PAO1. Recombinant PASP was proteolytic, with a zymogram mass of 50 kDa. PASP purified from PA103 produced extensive corneal epithelial erosions, whereas heat-inactivated PASP produced no erosions.


CONCLUSIONS
PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.
Marquart
Mary E
ME
Department of Microbiology, University of Mississippi Medical Center, Jackson, 39216, USA.
Caballero
Armando R
AR
Chomnawang
Mullika
M
Thibodeaux
Brett A
BA
Twining
Sally S
SS
O'Callaghan
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003315:Cornea / Q000187:drug effects* / Q000473:pathology; D003316:Corneal Diseases / Q000139:chemically induced* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011817:Rabbits; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C038495:Pseudomonas serine proteinase; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa