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1 Department of Molecular and
Cell Biology,
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-µm) or large (2.0-µm) pores. High transepithelial resistance
(Rt) Calu-3 and
cultured bovine tracheal monolayers
(Rt > 1,000 
· cm2)
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
Rt 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
Rt epithelia, low
Rt CFT1
(Rt = 100-200
· cm2) 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 Rt than in
high Rt 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
Rt CFT1 cells.
epithelial cells; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; green fluorescence protein; Calu-3 cells; trachea; epithelial polarity
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