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The N‐terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function
Author(s) -
Dasanayake Dayal,
Richaud Ma,
Cyr Normand,
CaballeroFranco Celia,
Pittroff Sabrina,
Finn Ron M.,
Ausió Juan,
Luo Wensheng,
Donnenberg Michael S.,
Jardim Armando
Publication year - 2011
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2011.07727.x
Subject(s) - biology , escherichia coli , vesicle , secretion , cytosol , biophysics , biochemistry , microbiology and biotechnology , membrane , gene , enzyme
Summary Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe‐like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N‐terminal segment of EspD, encompassing residues 1–171, contains two amphipathic domains spanning residues 24–41 and 66–83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His 6 ‐EspD 1–171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His 6 ‐EspD 1–171 undergoes a pH depended conformational change that increases the α‐helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp 47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His 6 ‐EspD 1–171 forms a homodimer that is postulated to promote EspD–EspD oligomerization and pore formation. Complementation of Δ espD null mutant bacteria with an espDΔ66–83 gene showed that this protein was secreted but non‐functional.