Research Article: Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis

Date Published: December 12, 2016

Publisher: Public Library of Science

Author(s): Ramyavardhanee Chandrasekaran, Anne K. Kenworthy, D. Borden Lacy, Steven R. Blanke.


Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.

Partial Text

Clostridium difficile, a gram-positive, spore-forming anaerobe, is the most common cause of healthcare-associated infections and gastroenteritis-associated death in the United States [1–3]. The pathogenesis of C. difficile is mediated by two large homologous exotoxins, TcdA and TcdB (308 kDa and 270 kDa, respectively), capable of causing epithelial cell death, fluid secretion and inflammation [4]. Recent studies, using isogenic single and double toxin knockout strains, have shown that either TcdA or TcdB alone can cause disease in animal models, with TcdB linked to severe disease phenotypes [5–7]. Most pathogenic isolates produce TcdA and TcdB emphasizing the need to consider both toxins when developing C. difficile therapeutics [8, 9].

TcdA and TcdB are the key virulence factors that mediate the pathology associated with C. difficile infection [5, 6]. Cellular intoxication by TcdA and TcdB depends on endocytosis and transport to acidified endosomal compartments within cells [18, 22, 23, 58]. Since these toxins represent excellent targets for therapeutic intervention, understanding the mechanism of toxin entry is a significant priority.




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