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 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 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., 1994) M cells has previously been documented. In the former instance, an upregulation of 9- to 40-fold higher translocation in co-cultures vs. controls was recorded. For V. cholerae possessing cholera toxin (ctx+), a sixfold increase in bacterial translocation was observed between M cell-like and Caco-2 cells (Blanco & DiRita, 2006). While a direct comparison of the V. cholerae and V. parahaemolyticus data is not possible due to differing experimental conditions (e.g. moi = 80 and 5, respectively), the increase is similar between the species. The eightfold increase in V. parahaemolyticus translocation between the 1- and 2-h time points is also reflective of the situation in V. cholerae, where a 13-fold increase was observed. Interestingly, unlike the ctx+ strain, ctx− V. cholerae did not cause a drop in TER, and furthermore, translocation was much reduced and did not increase between 1 and 2 h. We have shown here that translocation of V. parahaemolyticus coincides with TER disruption. The proteins responsible for the translocation and TER disruption upon V. parahaemolyticus infection of M-like cells remain to be identified, but as this Vibrio species does not possess cholera toxin, a different mechanism must be responsible. After 1 h of co-incubation, inhibition of the ERK signalling pathway and inactivation of TTSS-2 both reduced translocation of the bacteria across the co-culture model. However, during the later stages of infection, translocation was a TTSS-independent process that did not require MAPK activation. This is similar to the TTSS independence of Salmonella translocation across M cells (Martinez-Argudo & Jepson, 2008), but contrary to the translocation inhibition action of the E. coli TTSS (Martinez-Argudo et al., 2007), illustrating the unique attributes of each TTSS and their specialisation to the pathogenicity of each bacterial species. In conclusion, translocation of V. parahaemolyticus across the co-culture M cell-like model occurs in significant numbers and coincides with TER disruption.