Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD |
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Authors: | Abhishek Chatterjee Celia Caballero-Franco Dannika Bakker Stephanie Totten Armando Jardim |
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Affiliation: | From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada |
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Abstract: | Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli 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. The type III secretion system 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 cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits. |
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Keywords: | bacterial pathogenesis Escherichia coli (E. coli) membrane protein protein translocation type III secretion system (T3SS) EHEC EPEC EspD |
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