Following 1 and 2 h of co-incubation, the ∆TTSS-2 strain displayed a 2.6- and 1.6-fold decrease in translocation, respectively, compared to wt, although these decreases were not statistically significant (Fig. 3). These data demonstrate that the TTSS do not inhibit V. parahaemolyticus translocation. Instead the bacteria are transported across the M cell-like co-culture model independently

of TTSS-1, while TTSS-2 has a modest enhancing effect on translocation at early stages of infection. To investigate whether V. parahaemolyticus translocates across the M cell-like co-culture model by disrupting the epithelial monolayer, the TER was measured in response to infection with wt, ∆TTSS-1 or ∆TTSS-2 bacteria. Measurement of the TER is one of the main ways to examine epithelial integrity Sirolimus AG-014699 concentration in vitro (Terres et al., 1998) as it represents the resistance to ion flow across the epithelial monolayer. Infection of the co-culture model with the wt bacteria resulted in a sharp decrease in TER 1 h postinfection with a further decrease observed 2 h postinfection (Fig. 4a). Similar decreases were detected for the ∆TTSS-1 and ∆TTSS-2 bacteria. Consequently, examination of the effects of V. parahaemolyticus on the TER of the M cell-like co-culture model indicates

that the disruption occurs independently of either TTSS-1 or TTSS-2. Infection of the Caco-2 monolayer with wt bacteria also resulted in a decrease in TER (Fig. 4b). Comparison of these data indicates that V. parahaemolyticus infection results in an increase in TER disruption in co-culture models when compared to Caco-2 monolayers. Although Phosphoglycerate kinase not statistically significant, the difference

in TER decrease between Caco-2 and co-cultures was detected consistently. To determine whether MAPK activation has a role in the effects elicited by the bacteria on the co-culture, disruption of the TER in response to wt infection in the presence of MAPK inhibitors was examined. There was minimal difference between untreated co-cultures and co-cultures treated with the MAPK inhibitors (Fig. 4c). These nominal differences demonstrate that MAPK activation is not necessary for the disruption of the co-culture model in response to V. parahaemolyticus infection. Comparison of Caco-2 monolayers with a co-culture M cell model in this study indicates that V. parahaemolyticus is translocated in increased numbers (threefold increase) across the co-culture model. In the intestine, Peyer’s patch M cells actively endocytose bacteria and other foreign material for delivery to underlying lymphocytes, and this intracellular translocation would be the principal explanation for the observed increases (Neutra et al., 1996; Siebers & Finlay, 1996; Wong et al., 2003; Jang et al., 2004; Brayden et al., 2005). Enhanced transport of other M cell tropic bacteria such as Salmonella across an in vitro co-culture model (Martinez-Argudo & Jepson, 2008) and invasion through murine Peyer’s patches (Jones et al.