Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif

Andrew G Bease,Elizabeth A Blackburn,Cosmin Chintoan-Uta,Shaun Webb,Robin L Cassady-Cain,Mark P Stevens

doi:10.1016/j.jmb.2021.167200

Andrew G Bease, Elizabeth A Blackburn + Show 4 more

Open Access

https://doi.org/10.1016/j.jmb.2021.167200

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Abstract

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.

Highlights

Gastrointestinal bacterial infections have a substantial impact on both human and animal health
In order to investigate the hypothesis that lymphostatin is processed in T cells, we analysed lysates of T cells that had been treated with recombinant LifA from enteropathogenic Escherichia coli (EPEC) E2348/69 by western blotting using a polyclonal antibody that is known to recognise multiple domains of the protein
If the cysteine protease domain (CPD) of lymphostatin mediates autocatalytic cleavage in a manner similar to the large clostridial toxins (LCTs), we predicted that this cleavage should yield a fragment of at least 100 kDa encompassing the N-terminal glycosyltransferase domain that we previously localised to residues 309–860,12 and another fragment of at least 265 kDa encompassing the rest of the molecule

Summary

Introduction

Gastrointestinal bacterial infections have a substantial impact on both human and animal health. We detected rLifAC1480A at the same relative to the diluent control over five independent position as WT rLifA by gel electrophoresis and replicates of the study) revealed that at increased western blotting with anti-LifA antibodies concentrations of bafilomycin A1 and chloroquine, (Figure S4(D)).

Results

Conclusion