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In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection
K A Kernacki1, J A Hobden, L D Hazlett
1Department of Immunology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Purpose:
To establish if active pseudomonal proteases are present in vivo during corneal infection with Pseudomonas aeruginosa and to determine if the mouse strains used in these and previous studies have the ability to mount a nonocular antibody response to the purified proteases because antibodies to the bacterial proteases were not detected previously during in vivo ocular infection.
Methods:
At certain times after corneal infection with P. aeruginosa, corneas were harvested and supernatants from the corneal homogenates were analyzed for proteolytic activity by zymography and immunoreactivity by immunoblotting. The efficiency of the extraction procedures used in these studies was determined by incubating uninfected corneal homogenates with the purified proteases. The resultant supernatants were analyzed for alkaline protease and elastase activity. Additionally, mice were immunized intraperitoneally with the purified proteases with and without adjuvant to determine if the animals could mount a nonocular antibody response.
Results:
Corneas infected with P. aeruginosa demonstrated the presence of alkaline protease, but not elastase, by the two methods examined. The kinetics of the in vivo alkaline protease response closely parallels previously reported bacterial clearance studies in that peak alkaline protease activity was detected in corneal tissue when peak bacterial numbers also were observed in the eye, and it was absent when the eyes were sterile or nearly sterile. In addition, C57BL/6J mice were capable of mounting a nonocular antibody response to microgram quantities of both proteases only in the presence of adjuvant.
Conclusions:
In the model described, enzymatically active alkaline protease, but not elastase, was demonstrated in corneal tissues during in vivo infection. Concentrations of these proteases were much lower than those required to stimulate an antibody response.
Insights
Active alkaline protease from Pseudomonas aeruginosa was detected in infected mouse corneas, but elastase was not. Mouse strains could mount an antibody response to these proteases only with adjuvant, suggesting low in vivo concentrations.
Area of Science:
- Ophthalmology
- Microbiology
- Immunology
Background:
- Pseudomonas aeruginosa is a significant cause of bacterial keratitis.
- Pseudomonal proteases are virulence factors, but their in vivo presence and immunogenicity during corneal infection are not fully understood.
Purpose of the Study:
- To detect active pseudomonal proteases in vivo during corneal infection.
- To assess the capacity of mouse strains to generate a non-corneal antibody response to these proteases.
Main Methods:
- Corneal infection model in mice with Pseudomonas aeruginosa.
- Analysis of corneal homogenates for proteolytic activity (zymography, immunoblotting).
- Intraperitoneal immunization of mice with purified proteases to test antibody response.
Main Results:
- Active alkaline protease was detected in infected corneas; elastase was not.
- Peak alkaline protease activity correlated with peak bacterial load.
- Mice mounted an antibody response to proteases only when administered with adjuvant.
Conclusions:
- Enzymatically active alkaline protease, not elastase, is present during Pseudomonas aeruginosa corneal infection in this model.
- Protease concentrations in vivo are likely too low to elicit a significant antibody response without adjuvant.