Again no specific binding is seen with the MBP control and binding is greatly reduced with MBP-IfpC337G, whilst the MBP-Ifp fusion protein binds to individual cells with significant levels of fluorescence visible. Of 50 cells examined ~40% showed MBP-Ifp adherence, with only ~15% showing MBP-IfpC337G adhesion. Of those showing MBP-IfpC337G adherence, fewer fluorescing spots were observed per cell compared to MBP-Ifp, and these spots were smaller. Figure 3 FACScan analysis of the binding of purified MBP-fusion proteins to HEp-2 cells. Cells were incubated with (A) MBP-Ifp,

(B) MBP-IfpC337G, (C) MBP or (D) PBS and binding was visualised with anti-MBP and anti-rabbit Alexafluor 488 antibodies. Figure 4 Binding of purified MBP-fusion proteins to HEp-2 cells. HSP inhibitor Cells were incubated with (A) MBP-Ifp, (B) MBP-IfpC337G, (C) MBP or (D) PBS and binding was visualised with anti-MBP and anti-rabbit Alexafluor 488 antibodies. Representative cells are shown and the 10 μm ruler is shown in red. Interestingly, this binding appears to be localised

to specific foci on the cell surface, rather than a random scattering of fluorescence across the entire cell surface. This suggests that the protein is binding to specific receptors on the cell surface which are localised in foci. In order to investigate if a putative receptor was localised in cholesterol and sphingolipid-enriched plasma membrane micro-domains (lipid rafts), we used co-localisation assays. In this instance the GPI-anchored protein CD59, which is known to localise RG-7388 cell line to these microdomains [39], was used as a marker for the position of the lipid rafts. Confocal microscopy revealed that there is co-localisation between CD59 and MBP-Ifp bound on the cell surface, indicating that there is a putative receptor for Ifp present within these lipid rafts (Figure 5A). However, as there is binding of MBP-Ifp which does not co-localise, and as invasin is known to bind to β1 integrin, co-localisation

between MBP-Ifp and β1 integrin was also investigated (Figure 5B). No co-localisation was observed between MBP-Ifp and β1 integrin. Figure 5 Fluorescence microscopy showing co-localisation of (A) CD59 and (B) β1 integrin with purified MBP-fusion proteins on HEp-2 SPTLC1 cells. Cells were incubated with MBP-Ifp or MBP-IfpC337G. MBP-fusion proteins were visualised with anti-Ifp and anti-rabbit Alexafluor 594 antibodies. CD59 was visualised with anti-CD59 and anti-mouse Alexafluor 488 antibodies. β1 integrin was visualised with anti-β1 integrin and anti-mouse Alexafluor 488 antibodies. Representative cells are shown. Adhesion and invasion assays In order to confirm the role of Ifp as an adhesin, we constructed an insertion mutant in the ifp coding sequence of Y. pseudotuberculosis strain IP32953 (IPΔIFP). For comparative purposes, we also constructed an insertion mutant in the inv gene (IPΔINV), and a double insertion mutant (IPΔIFPΔINV) in the same strain.