A BACTERIAL PROTEASE PERTURBS THE PARACELLULAR BARRIER FUNCTION OF TRANSPORTING EPITHELIAL MONOLAYERS IN CULTURE

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the e pithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas ela stase (PE) on the barrier function of epithelia by using Madin-Darby c anine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. I mmunostaining of selected proteins of the ZO and zonula adherens was u sed to explore the effects of PE on junctional proteins. PE-treated mo nolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated th at, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescen ce staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zo nula adherens, was unaffected by the enzyme. Elastases from porcine pa ncreas and human leukocytes with similar enzymatic activity (6 U/ml) d id not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithel ial monolayers, in part, by changing the cell architecture and alterin g at least one protein of the ZO.