Cysteine supplementation differentially regulates toxin production and macrophage responses in two Clostridioides difficile strains

Background Clostridioides difficile infection is driven largely by toxin-mediated intestinal inflammation, and macrophages are among the first immune cells to respond to the pathogen. Whether cysteine-conditioned C. difficile differs in its interactions with macrophages across strains remains unclear. Methods Two C. difficile strains (R20291 and CD12038) were cultured without (C0) or with 20 mM cysteine (C20), washed, and cocultured with RAW264.7 macrophages. Toxin expression and secretion, macrophage cytotoxicity, ROS, Annexin V-FITC/PI staining, inflammatory gene expression, and dual bacterial-host RNA-seq were analysed. Results In the CD12038 strain, cysE / cysK expression was higher in C0 than in C20 ( P < 0.001), whereas tcdA / tcdB expression and supernatant TcdB concentrations were higher in C20 ( P < 0.05); the R20291 strain showed the opposite pattern ( P < 0.01). After coculture, the CD12038 C20 group caused more severe cytopathic effects, with greater LDH release ( P = 0.007), higher ROS levels ( P = 0.014), more Annexin V-FITC/PI-positive cells, and higher Il1b and Tnf-α mRNA expression ( P < 0.05) than the C0 group; for R20291, the C0 group showed stronger cytotoxicity, greater ROS ( P = 0.043), and higher Il1b / Tnf-α mRNA induction (both P < 0.05). Prokaryotic transcriptome analysis showed that CD12038 C20 was enriched in oxidoreductase activity and iron-sulfur cluster binding pathways, whereas R20291 C20 was enriched in protein folding, peptidase activity, and heat-stress response pathways. Host transcriptome analysis showed that pathways related to leukocyte migration, cytokine activity, the inflammatory response, and the immune response were enriched in cocultured macrophages, and R20291 C20 versus C0 was additionally enriched in myeloid leukocyte migration and cell population proliferation pathways. Conclusions The two tested C. difficile strains responded in opposite directions to cysteine supplementation with respect to toxin production and RAW264.7 macrophage responses in vitro . These strain-pair-specific associations suggest that cysteine availability may modulate toxin production and host inflammatory gene expression in a strain-dependent manner.