BackgroundCytolethal distending toxin (CDT) is a critical virulence factor of Campylobacter jejuni, and it induces cell death and regulates inflammation response in human epithelial cells. Pyroptosis is an inflammatory form of programmed cell death (PCD), but whether it is involved in CDT-mediated cytotoxicity remains elusive.AimsThis study explores the role and mechanism of pyroptosis in CDT-mediated cytotoxicity.MethodsHCT116 and FHC cell lines were treated with CDT. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Western blotting was used to measure the expression of related proteins in the pathway, and cell morphology observation, annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay were performed to evaluate the occurrence of pyroptosis.ResultOur results show that C. jejuni CDT effectively induces pyroptosis in a dose- and time- dependent manner in human colonic epithelial cells owing to its DNase activity. Specific pyroptotic features including large bubbles emerging from plasma membrane and LDH release were observed upon CDT treatment. Moreover, CDT-induced pyroptosis involves the caspase-9/caspase-3 axis, which is followed by gasdermin E (GSDME) cleavage rather than gasdermin D (GSDMD). N-acetyl cysteine (NAC), a reactive oxygen species (ROS) inhibitor, attenuates the activation of caspase-9/3, the cleavage of GSDME and pyroptotic characteristic, therefore demonstrating ROS initiates pyroptotic signaling.ConclusionsWe first clarify a molecular mechanism that CDT induces pyroptosis via ROS/caspase-9/caspase-3/GSDME signaling. These findings provide a new insight on understanding of CDT-induced pathogenesis at the molecular level.

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Natural selection has shaped a wide range of lifespans across mammals, with a few long-lived species showing negligible signs of ageing. Approaches used to elucidate the genetic mechanisms underlying mammalian longevity usually involve phylogenetic selection tests on candidate genes, analyses of differential gene expression between age cohorts or species, and measuring age-related epigenetic changes. However, the link between gene duplication and evolution of mammalian longevity has not been widely investigated. Here, we explored the association between gene duplication and mammalian lifespan by analysing 287 human longevity-associated genes across 37 placental mammals. We estimated that the expansion rate of these genes is eight times higher than their contraction rate across these 37 species. Using phylogenetic approaches, we identified 43 genes whose duplication levels are significantly correlated with longevity quotients (FDR < 0.05). In particular, strong correlation observed for four genes (CREBBP, PIK3R1, HELLS, FOXM1) appears to be driven mainly by their high duplication levels in two ageing extremists, the naked mole rat (Heterocephalus glaber) and the greater mouse-eared bat (Myotis myotis). Further sequence and expression analyses suggest that the gene PIK3R1 may have undergone a convergent duplication event, whereby the similar region of its coding sequence was independently duplicated multiple times in both of these long-lived species. Collectively, this study identified several candidate genes whose duplications may underlie the extreme longevity in mammals, and highlighted the potential role of gene duplication in the evolution of mammalian long lifespans.