Mechanism of Pseudomonas aeruginosa Small Protease (PASP), a Corneal Virulence Factor.
Invest Ophthalmol Vis Sci
; 59(15): 5993-6002, 2018 12 03.
Article
en En
| MEDLINE
| ID: mdl-30572344
ABSTRACT
Purpose:
Pseudomonas aeruginosa is the leading cause of contact lens-associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity.Methods:
PASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects.Results:
PASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity.Conclusions:
PASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Pseudomonas aeruginosa
/
Infecciones por Pseudomonas
/
Serina Endopeptidasas
/
Infecciones Bacterianas del Ojo
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Factores de Virulencia
/
Queratitis
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Invest Ophthalmol Vis Sci
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos