The EspF protein is translocated into host cells by the type III secretion system of
enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF
sequences differ between EPEC and EHEC serotypes in terms of the number of
SH3-binding polyproline rich repeats and specific residues in these regions as well
as residues in the amino domain involved in cellular localization. In this study we
have compared the capacity of different espF alleles to inhibit: (i) bacterial
phagocytosis by macrophages; (ii) translocation through an M-cell co-culture
system; (iii) uptake by and translocation through cultured bovine epithelial cells.
The espFO157 allele was significantly less effective at inhibiting phagocytosis and
also had reduced capacity to inhibit E. coli translocation through a human-derived in
vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast,
espFO157 was the most effective allele at restricting bacterial uptake into and
translocation through primary epithelial cells cultured from the bovine terminal
rectum, the predominant colonisation site of EHEC O157 in cattle and a site
containing M-like cells. As functional differences could not be simply assigned to
variation in established interactions of EspF with Sorting Nexin 9 and N-WASP,
yeast-2-hybrid screening was used to identify additional host proteins that may
interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was
identified from this screen. Mad2L2 was then demonstrated to interact with EspF
variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays.
While Mad2L2 has been shown to be targeted by the non homologous Shigella
effector protein IpaB to limit epithelial cell turnover, we presume that EspF
interactions with this protein may indicate a similar function to promote EPEC and
EHEC colonization.
The final section of work addressed whether bacterial interactions can actually
induce M-cell differentiation on follicle-associated epithelium. The work focused on
bovine rectal primary cell cultures interacting with Salmonella enterica serovar
Typhimurium. The type III secreted protein, SopB, was required for Salmonella to:
III
(i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii)
transform a subset of epithelial cells to a cell type that phenotypically and
functionally resembles specialized antigen sampling M cells; (iii) induce RANKL
and downstream RelB dependent NFkB signaling. The work suggests that
Salmonella may induce this cellular transformation to promote its invasion and
colonization of intestinal mucosa
