Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD.
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.
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada.
Abhishek
A
Chatterjee
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada.
Celia
C
Caballero-Franco
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada.
Dannika
D
Bakker
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada.
Stephanie
S
Totten
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada armando.jardim@mcgill.ca.
Armando
A
Jardim
D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D029968:Escherichia coli Proteins; C474481:EspD protein, E coli; D011994:Recombinant Proteins
EHEC; EPEC; Escherichia coli (E. coli); EspD; bacterial pathogenesis; membrane protein; protein translocation; type III secretion system (T3SS)