Airway epithelial tight junctions and binding and cytotoxicity of Pseudomonas aeruginosa.
The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosa to apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells. Binding of noncytotoxic P. aeruginosa strain O1 was assessed with P. aeruginosa that expressed green fluorescent protein. Binding of cytotoxic P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters with small (0.45-micrometer) or large (2.0-micrometer) pores. High transepithelial resistance (R(t)) Calu-3 and cultured bovine tracheal monolayers (R(t) > 1,000 Omega. cm(2)) bound P. aeruginosa very infrequently (<1 P. aeruginosa/100 cells) at the apical membrane, but P. aeruginosa bound frequently to cells near "free edges" at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes. Wounded high R(t) epithelia showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P. aeruginosa, and dead and living cells near the wound bound P. aeruginosa similarly. Compared with high R(t) epithelia, low R(t) CFT1 (R(t) = 100-200 Omega. cm(2)) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P. aeruginosa binding throughout the monolayer. Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low R(t) than in high R(t) airway epithelia. Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide. P. aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low R(t) CFT1 cells.
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
A
A
Lee
D
D
Chow
B
B
Haus
W
W
Tseng
D
D
Evans
S
S
Fleiszig
G
G
Chandy
T
T
Machen
D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D002417:Cattle; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004847:Epithelial Cells / Q000502:physiology; D004848:Epithelium / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007425:Intracellular Membranes / Q000502:physiology; D008164:Luminescent Proteins / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000473:pathology / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D019108:Tight Junctions / Q000502:physiology*; D014132:Trachea / Q000166:cytology / Q000502:physiology*; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins