PSEUDOMONAS-AERUGINOSA AND EPITHELIAL PERMEABILITY - ROLE OF VIRULENCE FACTORS ELASTASE AND EXOTOXIN-A

Lung injury in bacterial infection is a multifactorial phenomenon that involves bacterial metabolites and host factors. Primary isolates of type II pneumocytes and established cultures of Madin-Darby canine kid ney (MDCK) cells were used to study effects of Pseudomonas aeruginosa exoproducts on epithelial paracellular permeability. The results indic ate that elastase (PE) and exotoxin A (Exo A) have different, but comp lementary, actions that diminish epithelial barrier function. We measu red transepithelial electrical resistance (TER) and permeability coeff icient for mannitol (Pm) across cell monolayers plated on tissue cultu re membranes. Application of 100 ng/ml of Exo A to the basal side decr eased TER from 1,405 +/- 106 to 462 +/- 50 ohm (Omega) and increased P m for mannitol 6-fold in 16 h (P < 0.05). Application of Exo A to the apical side did not affect either TER or Pm, In contrast, PE (6.5 U/ml ) applied either apically or basolaterally reduced TER to 353 +/- 66 O mega and increased Pm by 10-fold within 90 min (P < 0.05). The increas e in permeability correlated with the number of bacteria that traverse d the epithelial monolayers. Fluorescent staining and western immunobl ot analysis of toxin-treated cells showed that two tight junctional pr oteins, ZO-1 and ZO-2, were depleted in monolayers treated with enzyma tically active PE. The junctional proteins decreased in cells treated overnight with Exo A but were not depleted. Neither agent diminished c ell viability as measured by trypan blue staining or release of radioa ctivity from Cr-51-labeled cells. Elastase from P. aeruginosa thus see ms to increase alveolar epithelial permeability by damaging tight junc tion-associated proteins, Exo A, through its effect on protein synthes is, may render the cells unable to restore the junctional proteins and thus the functional junctions.