Surface layer protein A from hypervirulent Clostridioides difficile ribotype 001 can induce autophagy process in human intestinal epithelial cells

• Published in: Microbial pathogenesis 2022-08, Vol.169, p.105681-105681, Article 105681
• Main Authors: Amirkamali, Sahar, Azimirad, Masoumeh, Nasiri, Gelareh, Goudarzi, Hossein, Noori, Maryam, Yadegar, Abbas, Ghalavand, Zohreh, Zali, Mohammad Reza
• Format: Article
• Language: English
• Subjects: Autophagy; Caco-2 cells; Clostridioides difficile; LC3B; SlpA; Surface layer protein
• ISSN: 0882-4010; 1096-1208
• DOI: 10.1016/j.micpath.2022.105681

Clostridioides difficile is the leading cause of nosocomial diarrhea with high morbidity and mortality worldwide. C. difficile strains produce a crystalline surface layer protein A (SlpA), which is an absolute necessity for its pathogenesis. However, its pathogenic mechanisms and its pro-inflammatory behavior are not yet fully elucidated. Herein, we report for the first time that SlpA extracted from C. difficile can induce autophagy process in Caco-2 cells. SlpA protein was purified from two C. difficile strains (RT001 and ATCC 700075). The cell viability of Caco-2 cells after exposure with different concentrations (15, 20, 25 μg/mL) of SlpA at various time points (3, 6, 12, 24 h) was measured by MTT assay. Acridine orange staining was used to visualize the hypothetical acidic vesicular organelles. The gene expression of autophagy mediators including LC3B, Atg5, Atg16L, and Beclin-1 was determined by quantitative real-time PCR assay. Western blotting assay was used to detect the expression of LC3B protein. MTT assay showed that different concentrations of SlpA did not induce significant changes in the viability of Caco-2 cells. SlpA at concentration of 20 μg/mL enhanced the formation of acidic vesicular organelles in Caco-2 cells after 12 h of exposure. Moreover, SlpA treatment significantly increased the expression of autophagy-associated genes, and increased the expression of LC3B protein in Caco-2 cells. In conclusion, our study demonstrated that SlpA is capable to induce autophagy in intestinal epithelial cells. These findings reveal a novel mechanism for the pathogenesis of C. difficile mediated by its SLPs. •Extracted SlpA from C. difficile stimulated the formation of autophagic vacuoles in Caco-2 cells.•Expression of autophagy-related genes was enhanced in Caco-2 cells after exposure with SlpA.•The expression of LC3B protein was significantly induced after treatment of Caco-2 cells with SlpA.•SlpA extracted from C. difficile RT001 could induce autophagy in Caco-2 cells after 12 h.