title: Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A.
abstract: 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 kidney (MDCK) cells were used to study effects of Pseudomonas aeruginosa exoproducts on epithelial paracellular permeability. The results indicate that elastase (PE) and exotoxin A (Exo A) have different, but complementary, actions that diminish epithelial barrier function. We measured transepithelial electrical resistance (TER) and permeability coefficient for mannitol (Pm) across cell monolayers plated on tissue culture membranes. Application of 100 ng/ml of Exo A to the basal side decreased TER from 1,405 +/- 106 to 462 +/- 50 ohm (omega) and increased Pm 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 omega and increased Pm by 10-fold within 90 min (P < 0.05). The increase in permeability correlated with the number of bacteria that traversed the epithelial monolayers. Fluorescent staining and western immunoblot analysis of toxin-treated cells showed that two tight junctional proteins, ZO-1 and ZO-2, were depleted in monolayers treated with enzymatically active PE. The junctional proteins decreased in cells treated overnight with Exo A but were not depleted. Neither agent diminished cell viability as measured by trypan blue staining or release of radioactivity from 51 Cr-labeled cells. Elastase from P. aeruginosa thus seems to increase alveolar epithelial permeability by damaging tight junction-associated proteins. Exo A, through its effect on protein synthesis, may render the cells unable to restore the junctional proteins and thus the functional junctions.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at Tyler 75710, USA.
authors.forename: A O
authors.initials: AO
authors.lastname: Azghani
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001426:Bacterial Proteins / Q000493:pharmacokinetics*; D001427:Bacterial Toxins; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured / Q000187:drug effects; D002857:Chromium / Q000378:metabolism; D004285:Dogs; D017097:Electric Impedance; D004848:Epithelium / Q000737:chemistry / Q000378:metabolism / Q000382:microbiology; D005098:Exotoxins / Q000494:pharmacology*; D007686:Kidney Tubules, Distal / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis; D009504:Neutrophils / Q000737:chemistry; D010196:Pancreatic Elastase / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000472:pathogenicity*; D011650:Pulmonary Alveoli / Q000166:cytology; D019108:Tight Junctions / Q000737:chemistry; D014774:Virulence; D037521:Virulence Factors; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002857:Chromium; D005098:Exotoxins; D008565:Membrane Proteins; D010196:Pancreatic Elastase; D010750:Phosphoproteins; D037521:Virulence Factors; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D000097668:Pseudomonas aeruginosa Exotoxin A