Campylobacter jejuni Infection Is Associated With Cell-Cycle, Redox, and Metabolic Remodeling of Human Intestinal Epithelial Cells at Single-Cell Resolution

Campylobacter jejuni is the leading cause of bacterial enteric infections worldwide, including in the US. It is also a zoonotic pathogen that is transmitted by food and water, causing diarrhea and intestinal inflammation, and is responsible for high morbidity and mortality in young children, the elderly, and immunocompromised patients. During infection, C. jejuni profoundly perturbs intestinal epithelial physiology, leading to widespread mucosal damage and inflammatory response. However, the heterogeneity and coordination of host transcriptional responses remain incompletely defined. We performed single-cell RNA sequencing of Caco-2 intestinal epithelial cells from uninfected Control and C. jejuni-infected biological samples using 10x Genomics GEM-X Flex chemistry. Our dataset comprised 159,088 cells and 18,142 measured features. A Seurat workflow resolved 19 transcriptional states and revealed a highly reproducible condition-associated redistribution of clusters across biological replicates. Our study demonstrated that C. jejuni infection was associated with increased representation of G2/M-classified cells and coordinated induction of checkpoint and mitotic-spindle genes, including CDKN1A, WEE1, MAD2L1, BUB1B, PLK1, CDC20, CDK1, and UBE2C. Furthermore, ranked enrichment independently identified a C. jejuni-enriched Reactome mitotic spindle checkpoint program (NES ~1.67, FDR ~0.013), while Hallmark analysis identified strong TNF/NF-kB, hypoxia, G2M checkpoint, TGF-beta, glycolysis, apoptosis, p53, and mTORC1-associated programs. Infection was also associated with significantly decreased JDP2 and higher NOX1 and DUOX2 gene expression. Furthermore, scMetabolism/AUCell identified 20 KEGG metabolic pathways at FDR <0.05 in a balanced 10,000-cell analysis, with 12 lower and 8 higher in infection. Together, these data support a model in which C. jejuni dramatically restructures epithelial transcriptional states and is accompanied by mitotic-checkpoint, inflammatory/stress, redox, and metabolic remodeling.