Enteropathogenic E. Coli Induces Intestinal Barrier Dysfunction By Exploiting Novel N-Wasp-Mediated Cytoskeletal Junction Regulatory Activities

Several IBD-associated polymorphisms have been associated with genes encoding subunits of the Arp2/3 complex, which promotes the assembly of actin filaments. As the immediate upstream activator of the Arp complex, NWASP is poised to integrate multiple cytoskeletal regulatory pathways required for apical junction complex (AJC) stability and plasticity, and is also the target of bacterial effector proteins that enable important enteropathogens to disrupt intestinal barrier integrity. The EPEC effector protein EspF, which has been shown to disrupt intestinal barrier, specifically activates N-WASP and sorting nexin 9 (SNX9), a protein that directly interacts with N-WASP and is involved in coordinating endocytosis and endosomal sorting. To investigate the role of N-WASP in junction regulation and response to pathogens, we generated cell lines depleted of N-WASP and SNX9, and examined dynamic AJC function and the ability of EPEC to induce barrier dysfunction. Stable N-WASP (NWKD) and SNX9 knockdown (SNX9 KD) Caco-2 cell lines were generated using lentiviral constructs expressing shRNA. >90% depletion of N-WASP and SNX9 was confirmed by Western blot. Cells expressing nonsilencing siRNA for N-WASP or SNX9 were used as controls (WT). WT and KD cells were plated on 0.4 um Transwell filters and cultured over 21 days, with serial measurements of transepithelial electrical resistance (TER). Assessment of TER and immunofluorescence localization of SNX9 and junction molecules (occludin, ZO-1, E-cadherin) was performed in confluent Caco-2 monolayers at steady state and under conditions that induce TJ disassembly (calcium depletion). Fully confluent WT and NWKD cultures were infected with EPEC strain E2348/69, and TER serially assessed during the 12 hours post-infection. N-WASP knockdown led to decreased epithelial barrier integrity, reflected by lower maximal TER (14 days after plating: NWKD 153.8 Ωcm2 vs. WT 310.8 Ωcm2, P < 0.001). During calcium-switch, NWKD cells exhibited delayed translocation of occludin at 12 and 20 hours after calcium repletion, and a delay in ZO-1 localization to the AJC at 12 hours. During AJC disassembly induced by rapid removal of calcium, NWKD monolayers exhibited delayed loss of TER, reflecting loss of junction plasticity. By immunofluorescence, we found that SNX9 localized to tight junctions and co-localized with junction proteins, such as occludin, during AJC disassembly. Notably, in contrast to N-WASP knockdown, depletion of SNX9 potentiated epithelial barrier function (7 days after plating: SNX9 KD TER 748 Ωcm2 vs. WT 347.2 Ωcm2, P = 0.03). We hypothesized that EspF utilizes N-WASP to disrupt the AJC, and predicted that wild type EPEC should be attenuated in its ability to induce junction disruption in NWKD. Compared to WT controls, NWKD Caco-2 cells were resistant to EPEC-induced barrier dysfunction: four hours after infection, TER in WT cells decreased by 61.4% vs. 28.8% in NWKD monolayers (P < 0.01 compared to WT). N-WASP, the immediate upstream activator of the IBD-associated Arp2/3 complex, regulates intestinal epithelial barrier function and response to enteric pathogens. A complex of N-WASP and SNX9 specifically regulates the disassembly of intercellular junctions and may represent a future target of strategies for mitigating intestinal barrier defects central to IBD pathogenesis.