ABSTRACT
The cytolethal distending toxin B subunit (CdtB) induces significant cytotoxicity and inflammation in many cell types that are involved in the pathogenesis of postinfectious irritable bowel syndrome (PI-IBS). However, the underlying mechanisms remain unclear. This study tested the potential role of Rab small GTPase 5a (Rab5a) in the process. We tested mRNA and protein expression of proinflammatory cytokines (interleukin-1β [IL-1β] and IL-6) in THP-1 macrophages by quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs), respectively. In the primary colonic epithelial cells, Cdt treatment induced a CdtB-Rab5a-cellugyrin association. Rab5a silencing, by target small hairpin RNAs (shRNAs), largely inhibited CdtB-induced cytotoxicity and apoptosis in colon epithelial cells. CRISPR/Cas9-mediated Rab5a knockout also attenuated CdtB-induced colon epithelial cell death. Conversely, forced overexpression of Rab5a intensified CdtB-induced cytotoxicity. In THP-1 human macrophages, Rab5a shRNA or knockout significantly inhibited CdtB-induced mRNA expression and production of proinflammatory cytokines (IL-1β and IL-6). Rab5a depletion inhibited activation of nuclear factor-κB (NF-κB) and Jun N-terminal protein kinase (JNK) signaling in CdtB-treated THP-1 macrophages. Rab5a appears essential for CdtB-induced cytotoxicity in colonic epithelial cells and proinflammatory responses in THP-1 macrophages.
Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not
