Antisera for detection of CdtA, CdtB, CdtC, CRP, and HtrA, respectively, were used for the immunoblot analyses and representative results of repeated experiments are shown. Molecular weight markers are shown in the lane (MW) on the left. See materials and methods for details about the relative amount of the extracts used. From this data, we suggest that substantial amounts of the CDT proteins were translocated into the periplasm of the bacterial cells and from there may be included

in the OMVs that are being released from the bacterial cell surface. The CDT proteins might be enclosed in the OMVs In order to further assess the nature of the association between CDT proteins and OMVs, we performed a dissociation Belinostat cell line assay as described in Materials and Methods. As shown in Figure 7A the CDT protein was recovered with OMVs in the

pellet after treatment with NaCl, Na2CO3, Urea, or HEPES buffer pH 7.3. Upon SDS solubilization of the OMVs, however, the CDT proteins could not be detected in the pellet but instead the proteins were released and remained in the supernatant after the subsequent centrifugation (Figure 7A, lanes 4 & 9). These results suggested that CDT was intimately associated with the OMVs. Resistance to high concentration urea (8 M) and liberation after SDS solubilization indicated that the proteins were not merely present as protein aggregates, but were surrounded by a membrane. To verify whether or not the Hsp60 protein was directly associated with OMVs, we monitored its fate in the dissociation assay using small molecule library screening the same procedure as was done for CDT proteins. As shown in Figure 7B, the Hsp60 protein was partially released into the supernatant after treatment with Na2CO3 and SDS but not with Urea. However, most of Hsp60 remained in the pellet

even after SDS treatment (Figure 7B, lane 4). Perhaps the formation of protein aggregates after detergent treatment caused Hsp60 to be retained in the pellet. Nevertheless, our results show that CDT and Hsp60 were not associated with OMVs in a similar manner as judged by these assays and the immunoelectron microscopy analysis. Figure 7 Analyses of CDT dissociation from OMVs. (A) Resminostat Dissociation assays of CDT proteins associated with OMVs from C. jejuni. Samples of vesicles in 50 mM HEPES were treated for 60 min on ice in the presence of: NaCl (1 M), Na2CO3 (0.1 M), urea (8 M) or SDS 1%, respectively. The samples were then centrifuged and the resulting pellets (lanes 1-5) and supernatants (lanes 6-10) were analysed by SDS-PAGE and immunoblot analyses with anti-CdtA, anti-CdtB, and anti-CdtC antisera. (B) Dissociation assays of Hsp60 protein. Samples were treated as in (A), and the immunoblot analysis was done with anti-Hsp60 antiserum. We have also analyzed whether the CDT protein subunits are associated with OMVs in other C. jejuni strains. Tests with OMVs samples from the C.