Introduction
Translocation of intestinal bacteria into the mesenteric lymph nodes and ascites plays a central role in the development of spontaneous bacterial peritonitis (SBP), a severe complication of liver cirrhosis. Immunosuppression, bacterial dysbiosis and increased intestinal permeability favor bacterial translocation. We have shown that the mucus layer in the intestine of patients with liver cirrhosis is thinner, supporting direct contact of bacteria with epithelial cells. This leads to degradation of cell-junctions (Haderer et al., Gut 2022), tissue damage and cellular stress. p53 is an important regulator of cellular stress responses. Depending on its specific isoforms, p53 can perform different functions. We investigate the association between bacterially induced cellular stress and the expression of different p53 isoforms using a novel luciferase-based reporter system (Truong et al. Nat Cell Biol 2021).
Aims & Methods
To quantify p53 isoform expression, we integrated exon-specific reporter systems (EXSISERS) into TP53 exons 2, 4, and 7. This allows distinction among three primary isoform groups (full-length p53, Δ40p53, and oncogenic Δ133p53 and Δ160p53) in HCT116 colon carcinoma cells. Using split proteins facilitates the removal of luciferase from the resultant p53 polypeptide, thereby maintaining the integrity of the p53 protein isoforms. This method enables the determination of the cellular ratio of p53 protein isoforms. HCT116 EXSISERS cells were co-cultured with a reference strain of Escherichia coli (E. coli) O6:Hnt, as well as clinical isolates of E. coli Ont:H7, Ont:H41, Proteus mirabilis, and Klebsiella pneumonia obtained from ascites of patients with SBP, for up to 2 hours. Isoform expression was quantified by measuring luciferase activities at intervals of 15 minutes to 2 hours using specific substrates. In addition, qPCR analyses were performed. Further, p53 isoform expression was evaluated after co-incubation with the cytokine IL-8 (mimicking inflammatory conditions) or bacterial components (LPS, MDP, CpG).
Results
Co-incubation with bacteria at MOI 5 resulted in upregulation of the cell-death inducing △40p53 isoform expression within 15 minutes. Simultaneously, we observed a gradual reduction of △133p53 and △160p53, known for their anti-apoptotic and pro-oncogenic properties, over a period of 2 hours. Thus, after bacterial co-culture isoforms inducing cell death or cell cycle arrest were expressed at a constant level, while oncogenic isoforms were downregulated. Interestingly, stimulation with CpG motifs of bacterial DNA interacting with TLR9, resulted in an upregulation of △40p53 and a reduced expression of △133p53 and △160p53 similar to that observed after bacterial co-incubation. In contrast, bacterial co-culture of EXSISERS cells with IL-8, LPS or MDP resulted in a downregulation of Δ40p53 isoform expression after 15 minutes.
Conclusion
p53 plays a central role in SBP pathogenesis. Direct contact with bacteria induces the pro-apoptotic isoform △40p53 to eliminate damaged cells, while constant translation of full-length p53 triggers a cell cycle arrest allowing epithelial cells to adapt to bacterial stress. Thus, in SBP, there is a delicate balance between cell survival and cell death in the intestinal epithelium. While △40p53 remained unaffected by IL-8, CpG motifs of bacterial DNA induced △40p53 upregulation. Further studies will decipher the signaling pathways and interactions leading to Δ40p53 activation of in order to develop targeted therapeutic approaches to protect the epithelial barrier.
References
Haderer M, Neubert P, Rinner E, et al. Gut 2022;71:580–592.
Truong, DJ.J., Phlairaharn, T., Eßwein, B. et al. Nat Cell Biol 2021;23:652–663.