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
