Background & 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.
Keywords: 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.