Lymphostatin is a large protein produced by pathogenic E. coli that inhibits the mitogen- and antigen-activated proliferation of T lymphocytes and synthesis of pro-inflammatory cytokines1. It is required for colonisation of cattle by Shiga toxin-producing E. coli and the virulence of enteropathogenic E. coli that cause diarrhoea in animals and humans. The activity of lymphostatin is dependent on a glycosyltransferase motif2 and a cysteine protease domain that mediates autocatalytic cleavage of the protein in lymphocytes3. The specific cellular targets of lymphostatin are unknown however recent data suggest that it acts proximal to T cell receptor activation and arrests the cell cycle. While lymphostatin can block lymphocyte proliferation in the femtomolar range in vitro1, the extent to which it inhibits the function of lymphocytes in vivo and the induction of adaptive immune responses to pathogenic E. coli are unknown. The proposed project aims to:
1. Define the uptake and cellular tropism of lymphostatin in the intestines.
2. Define the impact lymphostatin has on lymphocytes in the gut epithelium, draining lymph nodes and periphery.
3. Define if lymphostatin inhibits the induction of humoral and cell-mediated responses to itself and model antigens, and the role of catalytic motifs in these processes.
4. Identify cellular targets of lymphostatin and its mode of action.
To meet these objectives, we have developed strategies to express and purify native and mutated lymphostatin1-3, assays of its activity against lymphocytes1 and engineered bacterial strains to express or lack the protein. Animal models are in place to study the activites of lymphostatin in vivo, whether using recombinant protein or using bacterial strains. Lymphostatin homologues exist in pathogenic E. coli1 and scope exists to determine if these act in similar or different ways. The project will provide diverse training in microbiology, immunology and molecular biology.
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