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Pseudomonas deficient in protease IV has significantly reduced corneal virulence
L S Engel1, J A Hobden, J M Moreau
1Department of Microbiology, LSU Eye Center, Louisiana State University Medical Center, School of Medicine, New Orleans, USA.
Purpose:
The role of protease IV in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated by comparing a mutant strain completely deficient in protease IV activity with its protease IV activity-producing parent.
Methods:
A protease IV-deficient Pseudomonas strain PA103-29::Tn9 was generated by mutagenesis of strain PA103-29, which produces protease IV, through transposon insertion. Protease IV activity was determined by a casein agar assay, zymography, and cleavage of the chromogenic substrate, Chromozym PL. Corneal virulence was evaluated by slit lamp examination and bacterial cultures in both a rabbit intrastromal model and a mouse topical model of keratitis.
Results:
The protease IV-deficient strain PA103-29::Tn9 had significantly reduced corneal virulence relative to its parent strain PA103-29 in both a rabbit intrastromal model and a mouse topical model of infection. In the rabbit model, ocular damage (slit lamp examination score) mediated by the parent strain was severe at 32 hours after infection, whereas damage mediated by the mutant was minimal at both 32 and 55 hours after infection. This difference in virulence was not a result of differences in growth in vivo, because both strains grew equally. In the mouse model, eyes inoculated with the protease IV-producing parent strain had significant corneal damage as early as 24 hours after infection, whereas the protease IV-deficient mutant strain produced no significant corneal damage during 6 days of infection.
Conclusions:
The ability to produce active protease IV was the determining factor in the severity of corneal virulence. Protease IV appears to mediate corneal virulence and should be considered as a target in the development of medications designed to minimize corneal damage during Pseudomonas keratitis.
Insights
Protease IV is crucial for Pseudomonas aeruginosa keratitis virulence. A mutant lacking protease IV showed significantly reduced corneal damage in rabbit and mouse models, indicating protease IV as a therapeutic target.
Area of Science:
- Ophthalmology
- Microbiology
- Infectious Diseases
Background:
- Pseudomonas aeruginosa is a common cause of bacterial keratitis.
- Protease IV is an extracellular enzyme produced by P. aeruginosa.
Purpose of the Study:
- To investigate the role of protease IV in the pathogenesis of P. aeruginosa keratitis.
- To compare the virulence of a protease IV-deficient mutant with its parent strain.
Main Methods:
- Generated a protease IV-deficient P. aeruginosa strain (PA103-29::Tn9) via transposon mutagenesis.
- Assessed protease IV activity using casein agar assay, zymography, and chromogenic substrate cleavage.
- Evaluated corneal virulence in rabbit intrastromal and mouse topical keratitis models.
Main Results:
- The protease IV-deficient strain exhibited significantly reduced corneal virulence in both rabbit and mouse models.
- Rabbit eyes infected with the parent strain showed severe damage, while the mutant caused minimal damage.
- Mouse eyes infected with the parent strain developed significant corneal damage early, whereas the mutant caused no significant damage over 6 days.
Conclusions:
- Protease IV production is a key determinant of corneal virulence in P. aeruginosa keratitis.
- Protease IV mediates corneal damage and represents a potential therapeutic target for Pseudomonas keratitis.