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

Robin L. Cassady-Cain, Elizabeth A. Blackburn, Husam Alsarraf, Emil Dedic, Andrew G. Bease, Bettina Böttcher, René Jørgensen, Martin Wear, Mark P. Stevens*

*Corresponding author

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review


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 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, although 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 toward identifying the nature and consequences of modifications of host cell factors.

TidsskriftJournal of Biological Chemistry
Udgave nummer11
Sider (fra-til)5803-5816
Antal sider14
StatusUdgivet - 11 mar. 2016
Udgivet eksterntJa

Bibliografisk note

Funding Information:
This work was supported, in whole or in part, by core strategic funding from Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/J004227/1 (to R. L. C. C. and M. P. S.), Wellcome Trust Multi-User Equipment Grant 101527/Z/13/Z and Wellcome-UoE ISSF award (to E. A. B., M. W., and B. B.), and the DFF-Sapere Aude Starting Grant from the Danish Council for Independent Research Grant 11-104831/FSS) (to H. A., E. D., and R. J.). The authors declare that they have no conflicts of interest with the contents of this article.

Publisher Copyright:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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