To authenticate the mutation, the level of cell lysis induced by the Δbuy XAV-939 VP1680 strain was determined by the LDH assay. Over a 4 h period the viability of the Caco-2 cells co-incubated with the Δvp1680 strain was comparable to the viability of cells co-incubated with the ΔvscN1 strain (at the 4 h time point the percentage cell lysis values were: WT – 52±8%; ΔvscN1 – 10±2%; Δvp1680 -8±1%) confirming that the VP1680 TTSS1 effector protein is the principle factor responsible for the cytotoxicity

of V. parahaemolyticus towards epithelial cells. Analysis of the morphology of selleckchem the cells co-incubated with the Δvp1680 bacteria showed that the cells were still attached to the substratum as a confluent monolayer, but appeared rounded and did not display evidence of tight junctions (Additional file 1, Figure S1). In contrast cells co-incubated with ΔvscN1 bacteria were indistinguishable from non-infected cells. This indicated that VP1686 [37, 38] was being the translocated into host cells by Δvp1680 bacteria and that TTSS1 was functional in this strain. Analysis of the ability of this Δvp1680 strain to induce MAPK activation in the Caco-2 and HeLa cells was performed by immunoblotting of the extracted proteins with anti-phospho-JNK, selleck -p38 and -ERK antibodies (Figure 2). In Caco-2 cells, the Δvp1680 strain lacked the ability to activate p38 and JNK to the extent seen with the WT, indicating that VP1680

was the TTSS1 effector required for activation of these two MAPK. While reduced ERK activation was observed with the Δvp1680 strain as compared to the WT, a conclusive resolution could not be drawn, due to the low overall fold increase in ERK activation. In HeLa cells a more pronounced decreased phosphorylation of ERK occurred in response to Δvp1680. In contrast, VP1680

was only partly responsible for activation of p38 and JNK, as just a slight reduction in phosphorylation was seen in cells co-incubated Carnitine dehydrogenase with the V. parahaemolyticus strain lacking this effector as compared to WT bacteria. As activation of the MAPK is not abolished when VP1680 is non-functional, this suggests that there is an alternative TTSS1 effector that can activate MAPK in HeLa cells, but not in Caco-2 cells Our results show that VP1680 is necessary for the activation of JNK and p38 in Caco-2 cells and that JNK is involved in the VP1680-dependent cytotoxicity of V. parahaemolyticus. These data together demonstrate that VP1680 is required for the ability of V. parahaemolyticus to be cytotoxic to epithelial cells, at least in part through activation of JNK. Both TTSS are involved in modulation of IL-8 secretion by intestinal epithelial cells in response to V. parahaemolyticus In response to pathogenic bacteria, intestinal epithelial cells produce a number of pro-inflammatory cytokines and chemokines, such as IL-8 which attracts neutrophils to the site of infection and can lead to inflammatory responses that may facilitate bacterial infection and colonisation [39–41].