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Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence
L S Engel1, J M Hill, J M Moreau
1Department of Microbiology, Immunology, and Parasitology, LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans, USA.
Purpose:
A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed.
Methods:
The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection.
Results:
Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81).
Conclusions:
The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.
Insights
Protease IV directly damages corneas and significantly enhances Pseudomonas virulence in a mouse model of keratitis. This study confirms protease IV
Area of Science:
- Ophthalmology
- Microbiology
- Infectious Diseases
Background:
- Pseudomonas aeruginosa is a major cause of bacterial keratitis.
- Protease IV is a secreted virulence factor of Pseudomonas aeruginosa.
- A mutant deficient in protease IV shows reduced virulence in experimental keratitis.
Purpose of the Study:
- To determine the corneal toxicity of purified protease IV.
- To evaluate the ability of protease IV to augment the virulence of protease-IV-deficient Pseudomonas.
- To elucidate the role of protease IV in the pathogenesis of bacterial keratitis.
Main Methods:
- Intrastromal injection of purified protease IV (20-200 ng) into mouse corneas.
- Infection of mouse eyes with protease-IV-deficient Pseudomonas (1000 CFU) plus active or heat-inactivated protease IV.
- Assessment of ocular disease via slit-lamp examination at multiple time points.
- Quantification of bacterial load (CFU) in corneas at 27 hours post-infection.
Main Results:
- Purified protease IV (50-200 ng) induced corneal epithelial defects within 3 hours.
- Protease IV significantly augmented the virulence of the protease-IV-deficient strain (P < 0.0001).
- Neither active nor heat-inactivated protease IV affected bacterial growth in the cornea.
Conclusions:
- Protease IV possesses direct corneal toxicity.
- Protease IV significantly contributes to Pseudomonas virulence in experimental keratitis.
- These findings highlight protease IV as a key virulence factor in bacterial keratitis.