Role of the Corneal Epithelial Basement Membrane in Ocular Defense against Pseudomonas aeruginosa

Abstract

ABSTRACT Pseudomonas aeruginosa can invade corneal epithelial cells and translocates multilayered corneal epithelia in vitro, but it does not penetrate the intact corneal epithelium in vivo. In healthy corneas, the epithelium is separated from the underlying stroma by a basement membrane containing extracellular matrix proteins and pores smaller than bacteria. Here we used in vivo and in vitro models to investigate the potential of the basement membrane to defend against P. aeruginosa . Transmission electron microscopy of infected mouse corneas in vivo showed penetration of the stroma by P. aeruginosa only where the basement membrane was visibly disrupted by scratch injury, suggesting that the intact basement membrane prevented penetration. This hypothesis was explored using an in vitro Matrigel Transwell model to mimic the corneal basement membrane. P. aeruginosa translocation of multilayered corneal epithelia grown on Matrigel was ∼100-fold lower than that of cells grown without Matrigel ( P < 0.005, t test). Matrigel did not increase transepithelial resistance. Matrigel-grown cells blocked translocation by a P. aeruginosa protease mutant. Without cells, Matrigel also reduced traversal of P. aeruginosa and the protease mutant. Fluorescence microscopy revealed a relative accumulation of bacteria at the superficial epithelium of cells grown on Matrigel at 3 h compared to cells grown on uncoated filters. By 5 h, bacteria accumulated beneath the cells, suggesting direct trapping by the Matrigel. These findings suggest that the basement membrane helps defend the cornea against infection via physical barrier effects and influences on the epithelium and that these roles could be compromised by P. aeruginosa proteases.