The translocation of T3SS effectors into host 3-Methyladenine solubility dmso cells is the most distinctive function of T3SSs. However, the secretion of T3SS effectors is thought to be a different event from translocation (Lee & Galan, 2004) because there are several examples of exceptions in which constructs of the amino-terminal signal sequence of T3SS effectors fused

with a reporter protein can be observed in the supernatant and not inside host cells (Lee & Galan, 2004). To exclude this possibility for VocC, a translocation assay using VopC fused with the catalytic domain of CyaA was performed (Sory et al., 1995). This assay determines whether a T3SS effector–CyaA fusion is injected into eukaryotic cells by measuring intracellular cAMP levels because the activity of the CyaA catalytic domain requires calmodulin within eukaryotic cells. The wild-type strain expressing the VopC–CyaA fusion induced a high level of cAMP inside Caco-2 cells, while the ∆vocC strain did not, producing levels similar to the negative control (the T3SS2-deficient strain, vcrD2) (Fig. 3).

These results indicate that VocC is necessary for not only the secretion of VopC but also its translocation into host cells. Another characteristic of T3SS chaperones is the ability to bind their cognate effector. As expected from the screening assay used to identify T3SS2-associated chaperones (Fig. 1), VocC appeared to bind VopC. In this binding experiment, purified proteins were used to observe direct binding between the chaperone and an effector. A pull-down assay was performed using purified GST–VocC (42.6 kDa) and poly-histidine-tagged VopC (VopC–HIS; 45.9 kDa). Crizotinib research buy As shown in Fig. 4, GST–VocC coprecipitated Nutlin-3 chemical structure with VopC–HIS, while GST alone as a negative control did not. Another presumable substrate

for VocC, VopT, fused with a poly-histidine tag showed similar binding results to GST–VocC (data not shown). In previous studies, the binding of T3SS-associated chaperones and effectors and the amino-terminal secretion signal of effectors were required for efficient secretion via the T3SS (Arnold et al., 2009). Therefore, it was important to identify the chaperone-binding domain and the amino-terminal secretion signal of VopC. First, we used a series of truncated VopC mutants fused with CyaA in a pull-down assay with GST–VocC. As shown in Fig. 5a, the domain covering at least 100 amino acids from the amino terminus of VopC was responsible for binding to VocC. Additional pull-down assays using VopC21–100–CyaA showed that VocC bound to the amino-terminal 21–100 amino acids of VopC (Fig. 5b). Next, we examined whether an amino-terminal secretion signal existed in VopC. A secretion assay using V. parahaemolyticus T3SS1-deficient strains (ΔvcrD1) expressing a series of truncated VopC mutants fused with CyaA was carried out to assess the specific secretion of VopC fusions through T3SS2 (Fig. 5c).