Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli.

The Journal of Biological Chemistry
Robin L Cassady-CainMark P Stevens

Abstract

Attaching and effacing Escherichia coli cause diarrhea 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, 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 and small-angle x-ray scattering. 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 ...Continue Reading

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Methods Mentioned

BETA
GTPases
electrophoresis
PCR
restriction digest
blood draws
flow cytometry
ion-exchange chromatography
X-ray
size exclusion chromatography
ion exchange chromatography

Software Mentioned

DAMAVER
PHYRE
ATSAS
FloJo
RELION
DAMMIF
CONTIN
ATSAS AUTOPOROD
IMAGIC
PSIPRED

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