Outer Membrane Vesicle shedding by P. aeruginosa is Induced by Tear Fluid or Lysozyme and Compromises Corneal Epithelial Defenses Against Bacterial Adhesion and Traversal.

Abstract

Purpose: P. aeruginosa, the leading cause of contact lens-related corneal infection, can produce outer membrane vesicles (OMVs) in response to different stimuli (e.g. antimicrobials). OMVs can cause intoxication of host cells or contribute to biofilm formation. Here, we examined whether tear fluid or its components could induce OMV production by P. aeruginosa, and the impact of OMV pre-exposure on corneal epithelial susceptibility to bacteria.

Methods: P. aeruginosa strain PAO1 was grown overnight on TSA plates (37°C), resuspended in PBS to ~10¹¹ cfu/ml, and treated with fresh human tear fluid, tear components, or control buffer for 1 h. OMVs were extracted by centrifugation, filtration of supernatants and ultracentrifugation. Pellets (intact OMVs) were resuspended in PBS, quantified and analyzed by SDS-PAGE and TEM. OMV effects on PAO1 adhesion to C57BL/6 mouse corneas were studied using confocal microscopy. Mouse eyeballs were rinsed with PBS, tissue-paper blotted to enable bacterial adhesion (or not blotted), preincubated for 1 h with either intact OMVs, sonicated OMVs (disrupted OMV control) or PBS, then inoculated with PAO1-GFP ~10¹¹ cfu/ml (5 h, 37°C). Images were acquired using 633 nm (corneal cells) and 488 nm (GFP) lasers. Z stacks (1.0 µm) were collected from ≥3 random fields/sample. Image J was used for 3-D image reconstruction. Impact of OMV pre-exposure on PAO1 traversal of Transwell filter-grown human telomerase-immortalized corneal epithelial cells (hTCEpi) was assessed using ~10⁶ cfu/ml PAO1 (4 h, 37°C), and viable counts of bacteria reaching the basal compartment.

Results: Exposure to human tear fluid or lysozyme greatly increased OMV production (observed by TEM, quantified by densitometry, 3.4 and 2.3-fold increase respectively). Pre-exposing mouse corneas to purified intact OMVs increased their susceptibility to bacteria adherence ~ 3-fold (p < 0.05, student’s t test). Priming human corneal epithelial cells with OMVs made them ~ 3-fold more susceptible to bacterial traversal.

Conclusions: Human tear fluid or lysozyme can stimulate OMV production by P. aeruginosa, OMVs can then prime the corneal epithelium for bacterial adhesion (mouse corneas ex vivo) and cellular traversal (human epithelium in vitro). The mechanisms involved, and contribution to pathogenesis of infection in vivo, remain to be determined.

Keywords: 664 pseudomonas • 433 bacterial disease • 482 cornea: epithelium