Pore Forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Abstract Enterohemorrhagic E. coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with EHEC involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system (T3SS). The T3SS protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ~2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde crosslinking and rate zonal centrifugation suggested that EspD in LUV membranes forms a ~280-320 kDa oligomeric structure consisting of ~6-7 subunits.