Abstract
BackgroundClostridioides (formerly Clostridium) difficile infection is the leading cause of antibiotic-associated colitis. Studies have demonstrated that C. difficile toxin A (TcdA) can cause apoptosis of many human cell types. The purpose of this study was to investigate the relationships among exposure to TcdA, the role of the receptor for the globular heads of C1q (gC1qR) gene and the underlying intracellular apoptotic mechanism in human colonic epithelial cells (NCM 460). In this study, gC1qR expression was examined using real-time polymerase chain reaction (PCR), western blotting and immunohistochemical staining. Cell viability was assessed by the water-soluble tetrazolium salt (WST-1) assay, and cell apoptosis was assessed by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Mitochondrial function was assessed based on reactive oxygen species (ROS) generation, changes in the mitochondrial membrane potential (ΔΨm) and the content of ATP.ResultsOur study demonstrated that increasing the concentration of TcdA from 10 ng/ml to 20 ng/ml inhibited cell viability and induced cell apoptosis (p < 0.01). Moreover, the TcdA-induced gC1qR expression and enhanced expression of gC1qR caused mitochondrial dysfunction (including production of ROS and decreases in the ΔΨm and the content of ATP) and cell apoptosis. However, silencing of the gC1qR gene reversed TcdA-induced cell apoptosis and mitochondrial dysfunction.ConclusionThese data support a mechanism by which gC1qR plays a crucial role in TcdA-induced apoptosis of human colonic epithelial cells in a mitochondria-dependent manner.
Highlights
Clostridioides difficile infection is the leading cause of antibiotic-associated colitis
The concentration of C. difficile toxin A (TcdA) increasing from 10 ng/ml to 20 ng/ml significantly decreased cell viability and apparently increased cell apoptosis when compared with the mock group (p < 0.01, p < 0.001,)
To clarify whether the Globular heads receptor of C1q (gC1qR) gene is required for TcdA to entry into cells and induce apoptosis, firstly, we measured the gC1qR expression in the presence of TcdA at 10 ng/ml, the mRNA and protein expression of gC1qR were analyzed by qRT-polymerase chain reaction (PCR) and western blot, respectively
Summary
Clostridioides (formerly Clostridium) difficile infection is the leading cause of antibiotic-associated colitis. Studies have demonstrated that C. difficile toxin A (TcdA) can cause apoptosis of many human cell types. In vitro studies have demonstrated that TcdA can cause apoptosis of many human cell types, including monocytes [10], HeLa cells [11], endothelial cells [12], and intestinal epithelial cells [13,14,15]. Zhang et al demonstrated that toxin A treatment results in a significant loss of viability and apoptosis in a neuronal cell line; this effect was found to depend on increased production of reactive oxygen species (ROS) and upregulation of p38 MAPK activity and p21Cip1/Waf expression [16]. The role of mitochondrial dysfunction in TcdA-induced human intestinal epithelial cell death has not been thoroughly investigated
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