Garber, JohnMallick, Emily M.Scanlon, Karen M.Turner, JerroldDonnenberg, Michael S.Leong, John M.Snapper, Scott2018-05-302018Garber, John J., Emily M. Mallick, Karen M. Scanlon, Jerrold R. Turner, Michael S. Donnenberg, John M. Leong, and Scott B. Snapper. 2018. “Attaching-and-Effacing Pathogens Exploit Junction Regulatory Activities of N-WASP and SNX9 to Disrupt the Intestinal Barrier.” Cellular and Molecular Gastroenterology and Hepatology 5 (3): 273-288. doi:10.1016/j.jcmgh.2017.11.015. http://dx.doi.org/10.1016/j.jcmgh.2017.11.015.http://nrs.harvard.edu/urn-3:HUL.InstRepos:37067845Background & Aims Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a key regulator of the actin cytoskeleton in epithelial tissues and is poised to mediate cytoskeletal-dependent aspects of apical junction complex (AJC) homeostasis. Attaching-and-effacing (AE) pathogens disrupt this homeostasis through translocation of the effector molecule early secreted antigenic target-6 (ESX)-1 secretion-associated protein F (EspF). Although the mechanisms underlying AJC disruption by EspF are unknown, EspF contains putative binding sites for N-WASP and the endocytic regulator sorting nexin 9 (SNX9). We hypothesized that N-WASP regulates AJC integrity and AE pathogens use EspF to induce junction disassembly through an N-WASP– and SNX9-dependent pathway. Methods: We analyzed mice with intestine-specific N-WASP deletion and generated cell lines with N-WASP and SNX9 depletion for dynamic functional assays. We generated EPEC and Citrobacter rodentium strains complemented with EspF bearing point mutations abolishing N-WASP and SNX9 binding to investigate the requirement for these interactions. Results: Mice lacking N-WASP in the intestinal epithelium showed spontaneously increased permeability, abnormal AJC morphology, and mislocalization of occludin. N-WASP depletion in epithelial cell lines led to impaired assembly and disassembly of tight junctions in response to changes in extracellular calcium. Cells lacking N-WASP or SNX9 supported actin pedestals and type III secretion, but were resistant to EPEC-induced AJC disassembly and loss of transepithelial resistance. We found that during in vivo infection with AE pathogens, EspF must bind both N-WASP and SNX9 to disrupt AJCs and induce intestinal barrier dysfunction. Conclusions: Overall, these studies show that N-WASP critically regulates AJC homeostasis, and the AE pathogen effector EspF specifically exploits both N-WASP and SNX9 to disrupt intestinal barrier integrity during infection.en-USN-WASPCytoskeletonJunction RegulationEspFADF, actin depolymerization factorAE, attaching-and-effacingAJ, adherens junctionAJC, apical junction complexArp, actin-related proteinCR,Crb, CrumbsDBS100, David B. Schauer 100EcoRI, E. coli RY13 IEHEC, enterohemorrhagicEM, electron microscopyEPEC, enteropathogenicEspF, early secreted antigenic target-6 (ESX)-1 secretion-associated protein FFITC, fluorescein isothiocyanateiNWKO, intestine Neural Wiskott-Aldrich Syndrome protein knockoutKO, knockoutN-WASP, Neural Wiskott-Aldrich Syndrome proteinNWKD, Neural Wiskott-Aldrich Syndrome protein knockdownPBS, phosphate-buffered salinePCR, polymerase chain reactionshRNA, short hairpin RNASNX9, sorting nexin 9SNX9KD, sorting nexin 9 knockdownTER, transepithelial electrical resistanceTir, translocated intimin receptorTJ, tight junctionZO-1, zonula occludens-1Journal Article2018-05-3010.1016/j.jcmgh.2017.11.015