Department of Microbiology and Physiological Systems
Biochemistry | Cellular and Molecular Physiology | Digestive System | Gastroenterology | Hepatology | Molecular Biology
Background and 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.
ADF, actin depolymerization factor, AE, attaching-and-effacing, AJ, adherens junction, AJC, apical junction complex, Arp, actin-related protein, CR, Citrobacter rodentium, Crb, Crumbs, Cytoskeleton, DBS100, David B. Schauer 100, EHEC, enterohemorrhagic Escherichia coli, EM, electron microscopy, EPEC, enteropathogenic Escherichia coli, EcoRI, E. coli RY13 I, EspF, EspF, early secreted antigenic target-6 (ESX)-1 secretion-associated protein F, FITC, fluorescein isothiocyanate, Junction Regulation, KO, knockout, N-WASP, N-WASP, Neural Wiskott-Aldrich Syndrome protein, NWKD, Neural Wiskott-Aldrich Syndrome protein knockdown, PBS, phosphate-buffered saline, PCR, polymerase chain reaction, SNX9, sorting nexin 9, SNX9KD, sorting nexin 9 knockdown, TER, transepithelial electrical resistance, TJ, tight junction, Tir, translocated intimin receptor, ZO-1, zonula occludens-1, iNWKO, intestine Neural Wiskott-Aldrich Syndrome protein knockout, shRNA, short hairpin RNA
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© 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. Under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
DOI of Published Version
Cell Mol Gastroenterol Hepatol. 2017 Dec 15;5(3):273-288. doi: 10.1016/j.jcmgh.2017.11.015. eCollection 2018 Mar. Link to article on publisher's site
Cellular and molecular gastroenterology and hepatology
Garber, John J.; Mallick, Emily M.; Scanlon, Karen M.; Turner, Jerrold R.; Donnenberg, Michael S.; Leong, John M.; and Snapper, Scott B., "Attaching-and-Effacing Pathogens Exploit Junction Regulatory Activities of N-WASP and SNX9 to Disrupt the Intestinal Barrier" (2017). Open Access Articles. 3411.
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.