Translocation of bacteria across both monolayers may also be occurring partly by an active invasion mechanism, and although this requires further investigation, it explains the relatively high number of bacteria translocated by Caco-2. Compared to viable bacteria, a severe reduction in transport

of heat-killed Salmonella was previously observed (Martinez-Argudo & Jepson, 2008), suggesting a role for bacterial-directed invasion in the translocation process. Previous studies have shown that V. parahaemolyticus activates the intracellular MAPK signalling pathways to exert its effects on host cells. As a result, we investigated the role of MAPK LY294002 molecular weight activation in the bacterial translocation across M cell-like co-cultures. Immunoblotting experiments demonstrated that the MAPK was endogenously activated in uninfected co-cultures and therefore no increased activation was observed upon infection with V. parahaemolyticus (data not shown).

To determine whether the MAPK pathways are involved in bacterial translocation across the co-culture model, cells were pretreated with MAPK inhibitors (15 μM SP600125, 40 μM PD98059 and 5 μM SB203580, which inhibit the JNK, p38 and ERK pathways, respectively) 2 h prior to infection and maintained throughout the experiment. Co-cultures treated with SP600125, PD98059 and SB203580 displayed 1.2-, 6.6- Obeticholic Acid and 2.0-fold decreases in translocation, respectively, 1 h postinfection (Fig. 2a). Two hour postinfection, co-cultures treated with SP600125 and PD98059 displayed a 1.3- and 1.7-fold decrease in translocation, respectively,

while cells treated with SB203580 displayed a 1.8-fold increase in bacterial translocation (Fig. 2b). Statistical analysis of the data concludes that only the differences observed between untreated wt-infected co-cultures and those-treated with the ERK pathway inhibitor at 1 h postinfection are significant. The ERK signalling pathway is one of the most important in eukaryotic cells with roles in cell proliferation, differentiation and survival. PD98059 specifically inhibits the phosphorylation of ERK by inhibiting the activity of upstream MEK1/2, with limited off-target effects Fossariinae (Davies et al., 2000). These data indicate that ERK activity plays a role in the translocation of V. parahaemolyticus across the co-culture model during the early stages of infection. Studies investigating enteropathogenic E. coli have demonstrated that the bacterial TTSS inhibit the translocation of the bacteria across co-cultures, therefore, the influence of V. parahaemolyticus TTSS on M cell-like co-culture translocation was investigated (Martinez-Argudo et al., 2007). Individual single TTSS mutants were employed as previous studies have indicated that each TTSS delivers unique effectors into the host cell and each mediates unique effects on the host cell and in vivo (Park et al., 2004a, b; Hiyoshi et al., 2010; Matlawska-Wasowska et al., 2010).