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(Investigative Ophthalmology and Visual Science. 2001;42:2347-2354.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Phospholipase A2 in Rabbit Tears: A Host Defense against Staphylococcus aureus

Judy M. Moreau1, Dalia O. Girgis1, Emma B. H. Hume1,2, Joseph J. Dajcs1, Megan S. Austin1 and Richard J. O’Callaghan1,3

From 1 Department of Microbiology, Immunology, and Parasitology, Louisiana State University (LSU) Health Sciences Center, and 3 Department of Ophthalmology, LSU Eye Center, New Orleans.

PURPOSE. This study analyzed rabbit tears for anti-staphylococcal activity, the role of phospholipase A2 (PLA2) in this reaction, and the ability of enzyme inhibitors to promote bacterial survival.

METHODS. Contact lenses with Staphylococcus aureus were applied to scarified rabbit eyes. The colony-forming units (CFU) per cornea or lens were determined and pathology was scored by slit-lamp examination (SLE). The bactericidal activity was measured by incubating bacteria with rabbit tears or PLA2 at 33° or 37°C. Radiolabeled S. aureus was incubated with PLA2 or tears to quantify the release of a membrane component that was identified by thin-layer chromatography. Inhibitors of these reactions were also analyzed.

RESULTS. Application of Staphylococcus, on contact lenses, to rabbit corneas resulted in bacterial killing and limited inflammation. Incubation of tears and bacteria (1:1; v/v) in tryptic soy broth at 33°C decreased CFU approximately 4 logs. Tears (>=30 µl) or PLA2 (>=30 U) incubated with bacteria in phosphate-buffered saline were bactericidal. PLA2 (>=0.2 U) or tears (>=2 µl) cleaved bacterial membranes, liberating arachidonic acid. Spermidine or tetracaine inhibited cleavage of bacterial membranes by tears or PLA2 and spermidine promoted bacterial survival and growth in tears. Tears (60 µl) killed >99% of the bacterial inoculum, whereas bacteria incubated in tears plus spermidine approximately doubled in number.

CONCLUSIONS. PLA2 in rabbit tears kills Staphylococcus by hydrolyzing bacterial membranes to release arachidonic acid. Spermidine and tetracaine inhibited PLA2 activity and spermidine protected Staphylococcus from PLA2 in rabbit tears.




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