Effect of 3D Caco-2 cell culture system on Virulence Heterogeneity and Complex Host Responses to Listeria monocytogenes Infection

L. monocytogenes is an important foodborne pathogen that first infects the intestinal epithelium. This study used human colon adenocarcinoma cells (Caco-2) to construct 2D monolayer models and 3D spheroid models. The results showed that Caco-2 cells could form stable 3D spheroids in low-adhesion culture dishes. Compared with the 2D model, the invasion efficiency of each strain in the 3D model was significantly reduced, tight junction protein ZO-1 remained intact, and excessive ROS bursts were not induced during infection. In both 2D and 3D models, ST87 and ST5 exhibited higher virulence levels, while ST9 and ST121 showed lower virulence levels. Adhesion and invasion rates of serogroup II strains were significantly higher than those of serogroups IIa and IIc, and lineage II strains were more virulent than lineage I strains. In the 2D model, clinical strains displayed higher virulence levels than food strains, whereas in the 3D model, no significant differences were observed between these two sources. Transcriptomic analysis showed that after L. monocytogenes infection, highly virulent strains were more likely to activate host cell feedback regulatory pathways; the 2D model mainly induced transcriptional suppression in host cells and activated related pathways including cell adhesion, glycosylation modification, and morphological remodeling; while the 3D model better preserved host transcriptome homeostasis, induced host defense mechanisms including oxidative stress regulation, protein folding, and DNA damage repair during the infection process. Overall, this study demonstrates that the 3D Caco-2 cell model has practical value for evaluating the virulence heterogeneity of L. monocytogenes and the corresponding host response in vitro.