Figure 6.

Mutations in N-WASP– and SNX9-binding regions of EspF attenuate EPEC-induced junction disruption in vitro. (A) Schematic of point mutations made in each PRR of EspF, rendering it unable to bind to N-WASP or SNX9. (B) WT EPEC induced robust actin pedestal formation, disruption of ZO-1 localization, and recruitment of ZO-1 to the tips of actin pedestals; in contrast, EPECΔespF/pEspF_NWBD and EPECΔespF/pEspF_SNX9BD were both attenuated in their ability to induce TJ disruption, with persistent junction localization of ZO-1 (arrowheads) in infected cells. (C) WT cells infected with EPEC expressing WT EspF showed a 61.4% reduction in TER at 4 hours after infection; in contrast, EPECΔespF/pEspFNWBD was only able to induce a 28.4% reduction in TER, and there was minimal change in TER at 4 hours in monolayers infected with EPECΔespF/pEspFSNX9BD (P < .01 for WT EPEC vs EPECΔespF/pEspFNWBD; P < .01 for WT EPEC vs EPECΔespF/pEspFSNX9BD). Scale bars: 10 μm.