Research On Pyroptosis Induced By Campylobacter Jejuni Cytolethal Distending Toxin In Epithelial Cells

Campylobacter jejuni is one of the main pathogens causing bacterial foodborne gastroenteritis all over the world,which is responsible for at least 96 million cases of enteric infection globally each year.Cytolethal distending toxin(CDT)is an exotoxin secreted by C.jejuni and a critical virulence factor of C.jejuni,but its specific pathogenesis is unclear.In this study,human colonic epithelial cell lines HCT116 and FHC were taken as the research objects.By constructing the CDT deletion strain of C.jejuni and expressing CDT recombinant protein,the pathogenicity of C.jejuni CDT to intestinal epithelial cells and its specific mechanism were explored at the cellular level by means of flow cytometry,CCK-8,comet assay,Western blot,immunofluorescence and CRISPR/Cas9 and so on,which provided a certain theoretical basis for revealing the pathogenic mechanism of C.jejuni.The research results mainly include the following three aspects:1.C.jejuni CDT is cytotoxicImmunofluorescence showed that the marker of DNA double strand break,γH2AX,were observed after HCT116 and FHC cells were treated with CDT holotoxin and bacterial lysates from C.jejuni wild type strain.But the treatment of individual subunit and bacterial lysates from C.jejuni mutant strain did not exhibit such effect,confirming that CDT can lead to DNA damage.The results of comet assay showed the markerly trailing in nucleus,suggesting DNA damage.The CCK-8 testing also showed that CDT holotoxin and bacterial lysates from C.jejuni wild type strain decreased the cell viability,indicating C.jejuni CDT is cytotoxic in HCT116 and FHC cells.2.C.jejuni CDT induces pyroptosis in colonic epithelial cellsBy detecting the relevant indexes of pyroptosis(morphology changes,the cleavage of GSDME/GSDMD,the release of LDH in culture medium and ratio of apoptosis/pyroptosis),it was found that HCT116 and FHC cells exhibited balloon-like bubbles emerging from plasma membrane(canonical features of pyroptosis),increased LDH release,enhanced ratio of apoptosis/pyroptosis,suggesting that CDT induces pyroptosis in HCT116 and FHC cells in a dose and time-dependent manner.In addition,through GSDME knockout and GSDMD knockdown,it was found that CDT-induced pyroptosis was inhibited after knockouting GSDME,while there was no significant difference after GSDMD knockdown,indicating that CDT induced GSDME dependent pyroptosis rather than GSDMD dependent pyroptosis.3.CDT induces pyroptosis via ROS/caspase-9/caspase-3/GSDME pathwayThe cleavage of GSDME and the characteristics of pyroptosis induced by CDT were diminished via inhibiting the activity of caspase-3 chemically using Z-DEVDFMK or knocking down the expression of caspase-3 using si RNA,suggesting that caspase-3 plays an essential role on GSDME cleavage and pyroptosis mediation.Moreover,the activation of caspase-9 rather than caspase-8 was detected after CDT stimulation.Knocking down caspase-9 could significantly inhibit GSDEM cleavage and the activation of caspase-3.In addition,CDT is able to increase the level of intracellular ROS significantly,and CDT-induced pyroptosis and activation of caspase-9/3 were inhibited after scavenging intracellular ROS,illustrating that CDT induces cell death through ROS/caspase-9/caspase-3/GSDME pathway.