A283
ENTEROPATHOGENIC E COLI (EPEC)-INDUCED EPITHELIAL HYPORESPONSIVENESS TO SECRETAGOGUES IS ASSOCIATED WITH DECREASED MEMBRANE LOCALIZATION OF CFTR
C Ohland, R DeVinney, W MacNaughton
University of Calgary, Calgary, AB
Aims: EPEC infection causes severe diarrhea in humans, leading to substantial infant mortality in developing countries. The mechanisms of infection are well characterized, including the formation of a type III secretion system (TTSS) to inject bacterial effector proteins into the host cell. However, little is known about how EPEC affects intestinal epithelial ion and water secretion. We hypothesized that EPEC decreases epithelial ion secretion via alteration of host signalling pathways.
Methods: Monolayers of the human intestinal cell line T84 were exposed to log phase EPEC at a multiplicity of infection of 100 for 4 h. The secretagogue-induced ion transport was measured by change in short circuit current of cells mounted in Ussing chambers. Protein levels of the cystic fibrosis transmembrane conductance regulator (CFTR) were measured by Western blot and densitometry. Apical membrane localization of CFTR was determined by cell surface protein biotinylation followed by Western blot.
Results: Exposure of T84 cells to EPEC for 4 h decreased (p<0.05) the response to the cAMP-dependent secretagogue forskolin (control, 124.7±7.2; EPEC, 81.1±14.1 µAmp/cm2), without altering Ca2+-dependent secretion. Exposure to an EPEC mutant lacking the TTSS ATPase or either bacterial pore protein (ΔescN, or ΔescC and ΔescV, respectively) caused similar hyposecretion, indicating that translocation of effector proteins was not involved. Interestingly, EPEC mutants lacking the TTSS filament proteins (EspA, B or D) caused a partial reversal of the hyporesponsiveness to forskolin. This suggests that EspA, B and D are interacting with the epithelial cell independent of a functional TTSS in an unknown manner to cause decreased responsiveness to forskolin.
We next investigated whether epithelial ion channel expression or localization was altered by EPEC to decrease chloride secretion. While total CFTR protein was increased, cell surface expression was significantly decreased (64.1% of uninfected control) after exposure to EPEC. As the ΔescN mutant did not induce cell depolarization (as shown by basolateral transporters being biotinylated on the apical surface), but still decreased ion secretion and apical membrane levels of CFTR, we conclude that CFTR is being mislocalized to the cytosol which results in hyposecretion. This may be due to decreased recycling of the channel to the apical membrane, rather than increased internalization, as inhibiting endocytosis with phenylarsine oxide did not reverse EPEC-induced hyposecretion.
Conclusions: In conclusion, EPEC decreases cAMP-dependent ion secretion of T84 cells by reducing levels of apical membrane CFTR, which is partially associated with TTSS filament protein expression.