We suggest that the vesicle associated release of CDT proteins is a common feature among C. jejuni strains. In this context it is selleck compound also relevant to mention that a recent proteomic study showed the CDT protein was found to be associated with OMVs derived from the pathogenic E. coli strain IHE3034 . OMV-associated CDT is biologically active CDTs constitute
a family of genetically related bacterial protein toxins able to stop the proliferation of many different cultured cell lines. The primary effect of the CDTs, regardless of their bacterial origin, is eukaryotic cell cycle selleck products arrest at the G2/M stage with resultant cessation of cell division . Since we could detect all CdtA, CdtB, and CdtC subunits in vesicle samples from C. jejuni strain 81-176, we decided to test whether the CDT complex was active in such preparations. Earlier studies described that a purified CdtB on its own had no effect on HeLa cells, but when it was combined with CdtA and CdtC the HeLa cells showed cell cycle arrest in the G2/M phase . Results from other studies also indicate that CdtB internalization is necessary for Alvocidib manufacturer toxicity . In their study, they demonstrated that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E. coli extract containing CdtA and CdtC
whereas CdtB alone had no effect on HeLa cells. However introduction of the CdtB polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action . In the present study we used a human ileocecum
carcinoma cell line (HCT8) instead of the HeLa cell line. We considered that for the analysis of C. jejuni infection, a cell line representing the intestinal epithelium might be more relevant. In order to analyze how cultured HCT8 cells were affected by OMVs containing CDT, the cells were treated with the vesicle samples obtained from the C. jejuni wild type strain 81-176 and from the cdtA mutant strain DS104 pheromone (Figure 8A). The CDT-containing vesicle preparations from strain 81-176 induced a distinct enlargement of the HCT8 cells (Figure 8A, panel C&D) that was not observed in case of vesicles from the cdtA::km mutant (Figure 8A, panel E&F). As a means to quantify the effect of the OMVs on cell cycle arrest we measured the incorporation of [3H]-labeled thymidine by the HCT8 cells that had been treated with OMVs. The thymidine incorporation data clearly indicated that OMVs with CDT caused cell cycle arrest and the level of incorporation was reduced to ca 20% when monitored after 48 h of incubation (Figure 8B). Figure 8 Analyses of biological activities of CDT. (A) Cytolethal distending effect by OMVs on HCT8 cells.