Mode of action of lymphostatin, a key virulence factor of attaching & effacing Escherichia coli
Bease, Andrew Graham
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Attaching and effacing Escherichia coli are significant diarrhoeal pathogens that can spread between humans or via animal reservoirs. One important virulence factor is a large multifunctional protein called lymphostatin (LifA), which has been reported to inhibit the mitogen-stimulated proliferation of lymphocytes and mediate adherence to epithelial cells. Mutants of Shiga toxin-producing E. coli lacking lifA are significantly impaired in their ability to colonise cattle. Little is known about the mode of action of LifA, however in silico analysis has identified a putative glycosyltransferase domain homologous to that of large clostridial toxins (LCTs). A shortened form of LifA has been shown to be Type III secreted, however it is not known if this is true for the full-length protein. Type III secretion assays using the prototype enteropathogenic E. coli strain E2348/69 and isogenic lifA and Type III secretion system mutants confirmed that LifA can be secreted through this transport system. Working in collaboration, I was also able to demonstrate that LifA can be purified in an active form that binds uridine diphosphate-N-Acetylglucosamine (UDP-GlcNAc) but not UDP-glucose. In order to probe the importance of a putative catalytic DXD motif within the glycosyltransferase domain, an in-frame DXD to AAA substitution mutant of full-length LifA was constructed. The ability of the purified wild-type and mutated protein to bind UDP sugars and inhibit bovine T cell proliferation were then examined. DXD-AAA substitution resulted in loss of binding of UDP-GlcNAc and the ability to inhibit mitogenic stimulation of bovine T cells, without obvious changes to the biophysical properties of the protein. Unlike LCTs, wild-type LifA did not appear to be directly cytotoxic to HeLa or Jurkat cells using a fluorescence-based assay for release of lactate dehydrogenase. Future studies will seek to define the cellular targets and consequences of GlcNAc modification by lymphostatin, as well as identifying other possible mechanisms of secretion and its ability to act as an adhesin.