Interaction of Clostridioides difficile Toxin B and the endolysosomal transport and autophagic flux

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Clostridioides difficile infections are associated with diarrhoeal diseases and in severe cases can cause pseudomembranous colitis and also even lead to death. The main pathogenicity factors are the two toxins TcdA and TcdB, which belong to the group of large clostridial glucosyltransferases. The toxins are autotransporter that enter target cells and inhibit Rho-GTPases through glucosylation. These important signalling proteins regulate the structure of the actin cytoskeleton. Additionally, a cytotoxic effect was described for TcdB, which is independent of the glucosyltransferase activity and is mediated via excessive production of reactive oxygen species. In the course of investigating the intracellular activity of the glucosyltransferase domain, we observed a delayed degradation of the toxin, which is associated with a negative influence on lysosome function and is also independent of the inhibition of Rho-GTPases. This TcdB-induced reduction in lysosome activity correlates with the accumulation of the autophagosome marker LC3B thereby indicating a reduced autophagic flux. This is a yet unknown effect of TcdB on intracellular endolysosomal transport and autophagy, presumably resulting in a more efficient release into the cytosol and reduced degradation of the pathogenic glucosyltransferase domain. The generation of specific toxin mutants will help to investigate the molecular mechanism of action leading to the inhibition of lysosome function. Accumulation of galectin-3 or -8 on lysosomes may indicate endomembrane damage and provide a possible clue to toxin-induced disruption of lysosome membrane integrity. Pharmacological inhibitors might help to reveal an involvement of calcium channel as it is the case for the early cytotoxic effect of TcdB. The L-type calcium channel blocker nifedipine is able to abolish this early cytotoxic effect. Although the TcdB-induced inhibition of lysosomes is not nifedipine-sensitive, an analogous interaction with other calcium channels cannot be excluded. Electron micrographs will support immunofluorescence analyses of lysosomes and autophagosomes and provide evidence for altered quality and quantity of these organelles, as well as information on the causality of lysosome dysfunction and autophagosome accumulation. Through the recombinant production and modification of a variety of clostridial glucosyltransferases and other toxins, we are able to discriminate between a toxin-specific effect and a general effect of this class of toxins. The knowledge gained helps to better understand the pathogenesis of C. difficile-associated diseases and their frequent recurrences.