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Biophysical characterization and activity of lymphostatin, a multifunctional virulence factor of attaching & effacing Escherichia coli

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Original languageEnglish
Pages (from-to)5803-5816
JournalJournal of Biological Chemistry
Issue number11
Early online date19 Jan 2016
Publication statusPublished - 11 Mar 2016


Attaching & effacing Escherichia coli cause diarrhoea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins (LCTs), which are retaining glycosyltransferases with a DXD motif involved in binding of a metal-ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably-transformed plasmid expression clone. We constructed a tightly-inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS). Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated that lymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, though other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap towards identifying the nature and consequences of modifications of host cell factors.

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