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Updated: Oct 17, 2026
Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection
Published on: January 8, 2020
A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient
P S Estrellas1, L G Alionte, J A Hobden
1Department of Immunology/Microbiology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
Purpose:
Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence.
Methods:
Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis.
Results:
In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue.
Conclusions:
These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.
Insights
Pseudomonas aeruginosa elastase A and protease IV are key virulence factors in corneal disease, unlike alkaline protease. Strain Paer1, causing mild infections, lacked these proteases, highlighting their role in pathogenesis.
Area of Science:
- Microbiology
- Ophthalmology
- Infectious Diseases
Background:
- Pseudomonas aeruginosa proteases are implicated as critical virulence factors in corneal disease pathogenesis.
- Distinct P. aeruginosa strains exhibit varying clinical disease profiles, necessitating investigation into their specific virulence mechanisms.
Purpose of the Study:
- To compare protease production and ocular virulence between P. aeruginosa strain Paer1 (from CLARE) and strain KEI 1025 (from corneal ulcer).
- To evaluate the role of specific proteases, including elastase A, elastase, protease IV, and alkaline protease, in P. aeruginosa-induced keratitis.
Main Methods:
- Protease production was assessed using colorimetric assays, gelatin zymography, and western blots.
- Elastase A activity was quantified via a staphylolytic assay.
- Ocular virulence was determined using a mouse scratch model of keratitis.
Main Results:
- Strain Paer1 demonstrated significantly lower production of active elastase A, elastase, and protease IV compared to strains KEI 1025 and ATCC 19660.
- While all strains produced active alkaline protease, only KEI 1025 and ATCC 19660 induced severe keratitis in mice; Paer1 caused mild, transient infections.
- Genetic restoration of elastase activity in Paer1 did not confer a virulent phenotype, and co-infection led to the displacement of Paer1.
Conclusions:
- P. aeruginosa elastase A and/or protease IV are crucial contributors to ocular virulence.
- Alkaline protease and elastase appear to play a lesser role in the pathogenesis of P. aeruginosa keratitis.
