Characterisation of the type III secretion effector NleF

Abstract

The human-specific enteropathogenic Escherichia coli and the mouse-restricted Citrobacter rodentium colonise the host intestinal mucosa via attaching and effacing lesions, inducing severe diarrhoeal disease and transmissible murine colonic hyperplasia, respectively. C. rodentium is widely used as an in vivo model for EPEC infection, sharing a highly homologous arsenal of virulence factors and a similar infection strategy with EPEC. EPEC and C. rodentium pathogenesis relies on the locus of enterocyte effacement-encoded type III secretion system and delivery of effectors into the host cytosol to subvert host cell signalling including: actin dynamics, cell trafficking and immune signalling. The role of many effectors during infection remains unclear. We sought to identify the function of the non-LEE-encoded effector protein F (NleF). We discovered that EPEC infection of polarised epithelial cells activated a caspase-4-dependent non-canonical inflammasome response leading to the processing and secretion of IL-18, which was counteracted in a T3SS- and NleF-dependent manner. EPEC NleF interacts with the p20 and p10 subunits of caspase-4, associating with the substrate-binding domain, to inhibit its proteolytic activity and downstream inflammasome activation. Infection of mice with a C. rodentium nleF mutant enhanced IL-18 secretion and revealed that NleF is essential for the inhibition of the caspase-1/11-dependent inflammasome and recruitment of neutrophils in vivo. We further report that ectopically expressed NleF activated NF-κB nuclear translocation and the up-regulation of the pro-inflammatory chemokine IL-8. Infection of HeLa with an EPEC nleF deletion mutant abolished the T3SS-dependent activation of NF-κB and the expression of IL-8. NleF was identified to act upstream to IκBα activation and could be inhibited by the TAB2/3 inhibitor NleE1. However the mechanism of the pro-inflammatory role of NleF remains unclear. These findings identify novel roles for the T3SS effector NleF, furthering our understanding of the infection strategy of EPEC and the host epithelial cell inflammasome response.