Figure 6 shows that there is a significant decrease in the level of adhesion of IPΔIFP compared to wild type (IPWT), which could be restored by complementation with the wild type ifp gene (IPΔIFPpIFP) (Figure 6A). The inv mutant did not show as great a reduction in adhesion as IPΔIFP, but the double mutant SC79 clinical trial showed comparable levels to the ifp single mutant. A significant decrease in invasion of IPΔIFP compared

to wild type is observed (Figure 6B). Both IPΔINV and IPΔIFPΔINV show significant decreases in invasion compared to wild type; however, it was beyond the sensitivity of this assay to determine slight differences between these two strains. The average ratio of intracellular:extracellular bacteria for each of the strains associated with the HEp-2 cells was as follows; IPWT 1:8; IPΔIFP 1:8; IPΔINV 1:176; IPΔIFPΔINV 1:141 and IPΔIFPpIFP 1:8. To determine the role of the virulence factors of the pYV in the adhesion and invasion still seen in these assays, the strains were cured of the pYV plasmid and the differential staining assay repeated (Figure 6C). Invasion levels were all below the sensitivity of this assay, but a significant difference was observed between wild type and IPΔIFP, IPΔINV CA4P cost and IPΔIFPΔINV for adhesion. Although the expression analysis suggested the invasin should not be expressed at the time point used for these experiments, as there

was a significant difference between wild type and inv mutants, presence of invasin was examined by western blot (Figure 6D). Invasin was found to still be present at 37°C although at a reduced level compared to 28°C 15 hour cultures. No invasin was observed in IPΔINV

and IPΔIFPΔINV which confirms the mutation of the inv gene in these strains. Figure 6 Adhesion and invasion of HEp-2 cells by wild type IP32953 and defined mutants. (A) adhesion, (B) invasion (C) adhesion with pYV cured strains, using differential staining assay. Wild type (IPWT) was compared to insertional mutants of ifp (IPΔIFP) and inv (IPΔINV), an ifp and inv double mutant (IPΔIFPΔINV) and an ifp mutant with complemented ifp (IPΔIFPpIFP), by setting IPWT values to 100%. Strains cured of pYV are marked with “”c”". Data was 17-DMAG (Alvespimycin) HCl pooled from assays performed in triplicate on at least three independent occasions with statistical analysis by unpaired t-test and statistically significant results designated by *. ** indicates p value of <0.005, *** indicates p value of <0.0005. (D) Presence of invasin at 28°C and 37°C after 15 hours incubation detected by western blotting with anti-invasin monoclonal antibody. Galleria model of infection Galleria mellonella has been used as an infection model for several bacterial pathogens because it possesses an innate immune system with structural and functional homology to the mammalian immune system.