The broad long term goal of this research is to understand the role of corneal cell invasion by bacteria in the pathogenesis of bacterial infectious keratitis due to Pseudomonas aeruginosa, and to develop strategies for treatment and/or prevention of this disease. Since P. aeruginosa is believed to be an extracellular pathogen, research into the pathogenesis of this disease has focused on bacterial adherence to the cornea, bacterial toxins, and the host inflammatory response. It has now been demonstrated that P. aeruginosa can invade corneal cells; during corneal infection and in various in vitro models. Intracellular bacteria can evade host defenses and some antibiotics, and could thus explain why P. aeruginosa keratitis is often persistent and difficult to treat.
The first aim of this proposal is to test whether corneal cell invasion by P. aeruginosa contributes to the development of infectious keratitis. The proposed methods involve identification of means to inhibit P. aeruginosa invasion of corneal cells, and then use of these methods to inhibit invasion whilst monitoring the effect on development of disease. P. aeruginosa cannot infect an intact healthy cornea and infection is most commonly associated with either corneal injury or contact lens wear. Thus, two different experimental models for studying P. aeruginosa corneal infection will be utilized; a murine model involving abrasion injury to the cornea, and a rabbit model for contact lens overwear. Levels of bacterial invasion into cells will be determined by gentamicin survival assays, and the severity of disease assessed by macroscopic examination of the cornea. Contact lens wear is the leading cause of P. aeruginosa corneal infection in humans. The second hypothesis to be tested is that following the introduction of P. aeruginosa into the eye, contact lens wear eventually increases the total number of intracellular bacteria on the cornea in comparison to eyes that do not wear lenses, by one or more of three mechanisms. These are: 1) that environmental conditions occurring under a contact lens which alter the metabolic function of corneal cells also alter the susceptibility of these cells to P. aeruginosa invasion; 2) that contact lens wear increases P. aeruginosa invasion of cells secondarily to increased bacterial adherence to these cells; and 3) that contact lens wear impedes the clearance of intracellular P. aeruginosa from the ocular surface by interfering with superficial epithelial cell exfoliation and/or by stagnation of cells under the contact lens. This hypothesis will be tested by: 1) examining the effect of environmental manipulations on P. aeruginosa entry into cultured corneal epithelial cells and whole mouse cornea in vitro; 2) comparing P. aeruginosa invasion into superficial corneal epithelial cells removed from human contact lens wearing and control subjects; and 3) evaluating whether intracellular bacteria are stagnated in the rabbit eye during contact lens wear. Infectious keratitis is the most serious complication of contact lens wear. Since contact lenses are most frequently worn in healthy eyes, even a low incidence of this serious disease is unacceptable. Factors that make certain patients more susceptible to infection and new approaches for the prevention and/or treatment of infection could be identified by the proposed studies.
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