718 Enhanced Cytoskeletal Association of Ezrin By ESPF Is Involved in EPEC-Mediated Suppression of NHE3 Activity

Abstract

Sodiumhydrogen exchanger 3 (NHE3) plays an important role in sodium and fluid absorption throughout the intestine. Our previous work showed that enteropathogenic Escherichia coli (EPEC) significantly decreases NHE3 activity in human intestinal epithelial Caco-2 cells and PS120 fibroblasts transfected with NHE3. Mutation of the translocated effector protein EspF (∆espF) prevented the change in NHE3 activity. The aim of this study was to determine the mechanism by which EspF reduces NHE3 activity. NHE3 activity was assayed as 22Na+ uptake utilizing NHE inhibitors EIPA and HOE-694. EspF has several effects on intestinal epithelial cells including induction of cell death, alteration of vesicle trafficking via interactions with sorting nexin 9 (SNX9) and enhancing the cytoskeletal association of ezrin. An EspF point mutant that prevents cell death, L16E was used to determine if this phenotype was related to the decrease in NHE3. Complementation of ∆espF with the L16E mutation restored the decrease in NHE3 activity suggesting that cell death is not the cause of reduced NHE3 activity (WT EPEC -35%, ∆espF +3%, L16E -29%, p<0.05, n=15, NHE3 activity as percent change from uninfected). EspF interacts with SNX9 via proline rich repeats (PRR) and alters vesicle trafficking. As NHE3 is regulated by apical recycling, we tested the effect of EspFD3, a strain harboring point mutations in each PRR that is impaired for SNX9 binding. EspF-D3 restored the decrease in NHE3 activity in a ∆espF mutant (WT EPEC -52%, ∆espF 0%, +espF -38%, +espF-D3 -45%, n=15). Thus, interference with vesicle trafficking is not involved in EspF-mediated modulation of NHE3 activity. We published previously that EspF enhances the cytoskeletal association of ezrin. Ezrin regulates NHE3 activity by binding directly to NHE3 or to NHERF1/2. Expression of the scaffolding proteins NHERF1 and NHERF2 in PS120 fibroblasts enhances the NHE3 response to EPEC (NHE3 only -36%, +NHERF1 -56%, +NHERF2 -65%, n=9 p<0.05). To determine if ezrin was involved in EPEC-induced decrease in NHE3 activity, the effect of expressing full-length versus dominant negative ezrin was studied. While Caco-2 cells expressing full-length ezrin exhibited the expected 2-fold decrease in NHE3 activity in response to EPEC infection, expression of dominant negative ezrin prevented this response causing a minor but not significant, increase in NHE3 activity (p<0.01, n=9). Our data suggest that EspF reduces NHE3 activity, at least in part, via its effects on ezrin. These events likely contribute to the diarrhea associated with EPEC infection.