Pseudomonas aeruginosa is an opportunistic pathogen capable of causing devastating corneal infections, particularly among users of soft extended-wear contact lenses. About 24 million people in the U.S. wear contact lenses for cosmetic correction of refractive errors; 1 in 4,000 users of daily-wear soft contact lenses and 1 in 350 users of extended-wear contact lenses will develop ulcerative keratitis. Despite a rigorous regimen of antimicrobial chemotherapy, P. aeruginosa-infected corneas risk perforation within 24-48 hr of disease onset. Infected corneas which do not progress to perforation are often severely scarred and require a corneal transplant to restore vision. The pathogenesis of P. aeruginosa keratitis arises from a sequence of events which includes: 1) bacterial adherence to and invasion of a compromised cornea with the subsequent release of toxic bacterial exoproteins, and 2) tissue damage originating from the host inflammatory response (primarily an influx of polymorphonuclear leukocytes). Stromal scarring and loss of visual acuity occur even if the bacteria are eliminated with antibiotics. An awareness of the mechanism(s) by which the bacterium attaches to the cornea as well as a knowledge of which exoproteins are responsible for mediating tissue damage is essential to the design of chemotherapeutic agents or regimens that could lessen the severity of the disease or even prevent development of P. aeruginosa keratitis. Therefore, the specific aims of the proposed research are to: 1) construct a series of isogenic mutants of an ocularly virulent strain of P. aeruginosa deficient in alkaline protease, elastase A, elastase B, al three exoproteases, exotoxin A, and exoenzyme S via allelic replacement, or alternatively with tranposon insertion mutagenesis; 2) determine the corneal binding and ocular infectivity of these mutants in vivo in a mouse model of keratitis and in vitro using ocular organ cultures, and 3) examine the therapeutic potential of treating P. aeruginosa keratitis with topically applied polyclonal antibodies to these exoproteins.
