确定的细菌联合体凸显致癌细菌对DNA甲基化和肿瘤发生的影响

BACKGROUND: Colorectal cancer (CRC) is second leading cause of deaths in the USA. Bacteria like E. coli harboring the carcinogenic island pks promote CRC in preclinical models. However, the impact of carcinogenic bacterial DNA methylation and CRC development is unknown. METHODS: A defined consortia of 13 bacterial isolates (C13) containing either E. coli low pks (C13 - DSMZ) or E. coli high pks (C13-UM149) were used in the study. Mice were administered 10⁸ CFU of the bacterial consortia via oral gavage. 2 weeks later, they received 2% Dextran sodium sulfate (DSS) for one week, and tissues were collected at week 7 for analysis. Stools, colonic tumors and matched normal mucosa were collected. Stool samples were subjected to 16S rRNA sequencing. Tumors and normal mucosa were subjected to reduced representation of bisulfite sequencing (RRBS) and RNA-seq analysis for methylation and gene expression analysis respectively. Western Blot (WB) and Immunofluorescence (IF) analysis were used to determine DNA damage and cell proliferation in IEC-6 cells. Immunohistochemistry (IHC) was performed on colonic tissues. RESULTS: IEC-6 cells exposed to E. coli DSMZ exhibited lower DNA damage (gammaH2AX expression), whereas cells treated with E. coli UM149 showed elevated gammaH2AX. Mice colonized with C13-UM149 developed significantly more tumors than those with C13-DSMZ (p = 0.046). IHC of colonic tissues showed that C13-UM149 exhibited higher Ki-67 (cell proliferation), beta-catenin (oncogenic signaling), and gammaH2AX compared with C13-DSMZ (p < 0.05). Genome-wide methylation analysis showed broader CpG alterations in C13-DSMZ tumors (247,797 CpGs) than C13-UM149 mice (172,727 CpGs). Compared to C13-DSMZ, C13-UM149 tumors had 70,650 hyper and 65,024 hypomethylated CpGs, and 192 hyper and 220 hypomethylated promoters. These changes were associated with upregulation of Il5ra (inflammation), Galr1 (tumor growth), and Minar1 (tissue remodeling), and downregulation of Tmed6 (protein trafficking) and Pcdhb19 (adhesion) in C13-UM149 tumors. In normal mucosa, C13-UM149 altered 28,316 hyper and 59,780 hypomethylated CpGs with increased Aldoart1 (glycolysis) and Palm3 (immune signaling) expression. C13-UM149 tumors also showed promoter hypomethylation driving increased expression of Sema4c (tumor invasion) and promoter hypermethylation accompanied by increased Mmp9 expression (matrix remodeling). C13-DSMZ tumors exhibited promoter hypermethylation with elevated expression of Mecom (tumor progression) and promoter hypomethylation with increased Wnt7b (oncogenic signaling). CONCLUSION: Distinct pks activities differentially modulate DNA methylation and influence gene expression and tumor development. Our findings suggest that the methylome can shift in two directions depending on the bacterial context, with predominant hypermethylation in tumors and hypomethylation in normal tissues.