Vibrio cholerae-induced cellular responses of polarized T84 intestinal epithelial cells are dependent on production of cholera toxin and the RTX toxin

Abstract

To study the utility of in vitro-polarized intestinal cell monolayers for modeling Vibrio cholerae-host cell interactions, we added live V. cholerae bacteria to the apical surfaces of polarized T84 cell monolayers and monitored changes in electrical properties. We found that both classical and El Tor strains produce cholera toxin after addition to the monolayer, but induction is most likely due to medium components rather than bacterium-cell interactions. We also found that the RTX toxin is produced by El Tor strains. This toxin caused a loss of the barrier function of the paracellular tight junction that was measured as a decrease in transepithelial resistance. This decrease occurred when bacteria were added to either the apical or basolateral surfaces, indicating that the RTX toxin receptor is expressed on both surfaces. These results are discussed with regard to the applicability of the polarized T84 cell monolayers as an in vitro model of host-pathogen interactions.

FIG. 1

Responses of T84 polarized monolayers to the addition of V. cholerae differ between classical and El Tor strains. PBS-washed classical strain O395 (A) and El Tor strain N16961 (B) were added to the apical chamber after the first three readings were recorded. Values are means and standard deviations of triplicates in a single experiment.

FIG. 2

Changes in TER after addition of V. cholerae El Tor bacteria are due to RTX toxin, not HA/protease. (A) V. cholerae Bah1 (HA/protease⁺ [HA/P⁺]) is compared to Bah1P (HA/P⁻). (B) Bah1P (RTX⁺) is compared to Bah2P (RTX⁻). Values were derived by dividing the recorded resistance (R) value by the mean of three initial resistance values recorded before addition of bacteria and are means and standard deviations of triplicates in a single experiment.

FIG. 3

RTX toxin is active when bacteria are added to either the apical or basolateral chamber. PBS-washed Bah1P (10 μl) was added to the apical chamber or Bah1P (50 μl) was added to the basolateral chamber. Both additions correspond to a 1:20 dilution in the total volume of the chamber. Values were derived by dividing the recorded resistance (R) value by the mean of three initial resistance values recorded before addition of bacteria and are means and standard deviations of triplicates in a single experiment.

FIG. 4

CT expression in El Tor V. cholerae. Both KFV82 (A) and KFV105 (B) have deletions in the rtx locus and in hapA eliminating factors that disrupt integrity of the paracellular tight junctions. The effect of CT expression on TER and Isc in a CT-positive strain (A) is shown in contrast to results with a ΔctxAB isogenic mutant (B). All values are means and standard deviations from triplicates in a single experiment.