EPEC Effector Protein EspF Disrupts Epithelial Polarity
Authors: Pastrana Rocio Tapia , Shan Kuangda , Kralicek Sarah E , Hecht Gail 
Published: November 2023

EPEC disruption of tight junctions (TJ) is dependent on type III secreted effectors, EspF, Map, and EspG, although data indicate their impact on TJs is not direct. The apical Crumbs (Crb) complex (Crumbs3, PATJ and Pals1) and the apical Par and lateral Scribble complexes are essential for epithelial cell polarization and TJ assembly. These complexes direct and retain proteins at the apical and basolateral membranes and TJs. We hypothesize that specific EPEC effector proteins perturb epithelial polarity complex proteins which in turn alters TJ structure and barrier function. Crb is a transmembrane protein and determinant of apical‐basal cell polarity in epithelial cells. Crb3 regulates TJ formation through its cytoplasmic domain and interaction with several cytosolic partners. The aim of this study was to determine the effect of EPEC on Crb3 and on the development of polarity. To determine the impact of EPEC infection on Crb3 expression and localization, western blot analysis and immunolocalization studies were performed on intestinal epithelial cells (SKCO‐15) infected with wild‐type (wt) EPEC. EPEC induced the movement of Crb3 from the membrane to the cytoplasm by 1–2 hrs without altering the level of expression. The EPEC effector EspF, is largely responsible for the perturbation of TJs associated with EPEC infection. Crb3 localization was therefore asessed in SKCO‐15 cells infected with an EPEC espF deletion mutant (ΔespF). Deletion of espF attenuated the loss of Crb3 from the membrane. In order to specifically investigate the impact of EspF on Crb3, EspF‐GFP was expressed in MDCKII and SKCO‐15 monolayers and Crb3 localization evaluated. EspF‐GFP expression induced Crb3 internalization, suggesting that this effector directly contributes to Crb3 mislocalization. EspF has been reported to localize to host mitochondria and promote cell death. To determine if the alteration in Crb3 localization was due to apoptosis, cells were transfected with EspFL16E‐GFP, an EspF site‐directed mutant that ablates the apoptotic phenotype. Expression of EspFL16E also caused a shift of Crb3 from membrane to cytoplasm despite its inability to promote apoptosis. Polarity proteins are the main regulators of cyst morphogenesis in 3D culture. We therefore examined the effect of EspF expression on 3D cyst formation in MDCKII cells. Expression of EspF‐GFP interfered with cyst development. Instead of developing organized, single lumen cysts, EspF caused the formation of multi‐lumen cysts. EspFL16E‐GFP also resulted in the development‐of multi‐lumen cysts, suggesting that the effect of EspF on the development of polarity is not due to increased apoptosis. To determine if the interference of EspF on polarized cyst formation correlated with alterations in TJ barrier function, an inducible Tet‐On system for expressing EspF‐HA in SKCO‐15 cells was generated. EspF‐HA was induced by the addition of doxycycline (+dox) for 3 days. Control monolayers show no difference in TER after the addition of doxycycline. In contrast, monolayers expressing EspF‐HA (+dox) exhibited a significant decrease in TER as compared to non‐induced cells (‐dox) (−49±9% vs +16±9% change from baseline, respectively; n=3, *p=0.01). These data support the hypothesis that EPEC‐induced perturbation of TJ structure and function is a downstream consequence of disruption of epithelial polarity complexes and that EspF contributes to this phenotype.