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

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.
Author Summary Clostridium difficile is a bacterial pathogen that causes nearly half a million infections each year in the United States. It infects the human colon and causes diarrhea, colitis and, in some cases, death. C. difficile infection is mediated by the action of two large homologous toxins, TcdA and TcdB. Disruption of host cell function by these toxins requires entry into cells. There are multiple ways for pathogens and virulence factors such as viruses and toxins to enter host cells. The entry mechanism is often directed by a cell surface receptor and can impact the trafficking and virulence properties of the pathogenic factor. Investigating the internalization strategy can provide critical insight into the mechanism of action for specific pathogens and virulence factors. In our current study, we sought to determine the strategy utilized by TcdA to enter host cells. We show that TcdA uptake occurs by a clathrin- and caveolae-independent endocytic mechanism that is mediated by PACSIN2 and dynamin. We also show that TcdA and TcdB can utilize different routes of entry, which may have implications regarding their cytotoxic mechanisms. In summary, our results provide new insights into the mechanism of cellular intoxication by TcdA and the role of PACSIN2 in endocytosis.