Pseudomonas aeruginosa causes a devastating form of keratitis that is typically contact lens associated. Progress in lens development to allow more oxygen penetration, according to new preliminary clinical studies, has not prevented Gram-negative bacterial keratitis. Studies of experimental Pseudomonas keratitis indicate that corneal damage is mediated by the action of bacterial proteins that, once released into the tissue, continue to act free of inhibition by antibiotics or other drugs now in existence. Of the multiple proteins produced by Pseudomonas, the ? one that has been best shown to be the """"""""aggressin"""""""" that mediates corneal damage is protease IV. This protease is only produced by P. aeruginosa and every strain tested to date produces this enzyme. Bacterial genetic experiments have demonstrated that the production of this enzyme provides substantial corneal virulence. ? Given the problem of Pseudomonas keratitis and the proven importance of bacterial proteins in the pathogenesis, a long-term goal is to develop a means to inhibit the aggressin proteins that damage the cornea once they are produced by intra-corneal bacteria. Toward that goal, the following specific aims are proposed: 1) develop an antibody capable of neutralizing protease IV and use this antibody as passive immunization to minimize corneal damage (this approach has been effective for Staphylococcus keratitis); 2) quantify the contribution of other proteases, including two uncharacterized enzymes, to corneal pathology to determine if they too should be targets for protective immunotherapy; 3) determine the host molecules, including host defense molecules, that are destroyed by bacterial proteins during infection; and 4) determine the importance of specific bacterial proteins to the host responses that typify Pseudomonas keratitis. These host responses to be analyzed include activation of matrix metalloproteinases, production of inflammatory cytokines, and increases in comeal calcium concentration. Critical to this research are the recent development of Pseudomonas putida strains that produce individual gene products of P. aeruginosa. These engineered strains can infect and replicate in the rabbit cornea without causing corneal damage unless the cloned genes of P. aeruginosa are produced. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
7R01EY012961-06
Application #
7061516
Study Section
Special Emphasis Panel (ZRG1-AED (01))
Program Officer
Shen, Grace L
Project Start
2000-05-01
Project End
2007-07-31
Budget Start
2005-05-16
Budget End
2005-07-31
Support Year
6
Fiscal Year
2004
Total Cost
$118,406
Indirect Cost
Name
University of Mississippi Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
928824473
City
Jackson
State
MS
Country
United States
Zip Code
39216
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Thibodeaux, Brett A; Caballero, Armando R; Marquart, Mary E et al. (2007) Corneal virulence of Pseudomonas aeruginosa elastase B and alkaline protease produced by Pseudomonas putida. Curr Eye Res 32:373-86
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Thibodeaux, Brett A; Caballero, Armando R; Dajcs, Joseph J et al. (2005) Pseudomonas aeruginosa protease IV: a corneal virulence factor of low immunogenicity. Ocul Immunol Inflamm 13:169-82
Marquart, Mary E; Caballero, Armando R; Chomnawang, Mullika et al. (2005) Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions. Invest Ophthalmol Vis Sci 46:3761-8
Marquart, Mary E; Dajcs, Joseph J; Caballero, Armando R et al. (2005) Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor. Med Microbiol Immunol 194:39-45
Thibodeaux, Brett A; Dajcs, Joseph J; Caballero, Armando R et al. (2004) Quantitative comparison of fluoroquinolone therapies of experimental gram-negative bacterial keratitis. Curr Eye Res 28:337-42
Caballero, Armando; Thibodeaux, Brett; Marquart, Mary et al. (2004) Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases. Invest Ophthalmol Vis Sci 45:522-30

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