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

艰难梭状芽胞杆菌感染与腹泻疾病有关，严重时可引起伪膜性结肠炎，甚至导致死亡。主要致病因子为TcdA和TcdB两种毒素，属于大型梭状芽胞杆菌葡萄糖基转移酶。这些毒素是进入靶细胞并通过糖基化抑制Rho-GTP酶的自身转运蛋白。这些重要的信号蛋白调节肌动蛋白细胞骨架的结构。此外，还描述了TcdB的细胞毒性作用，这种作用独立于葡萄糖转移酶的活性，并通过过量产生活性氧来调节。在研究葡萄糖转移酶结构域细胞内活性的过程中，我们观察到毒素的延迟降解，这与溶酶体功能的负面影响有关，也与Rho-GTP酶的抑制无关。这种TcdB诱导的溶酶体活性降低与自噬标记LC3B的积累相关，从而表明自噬通量减少。TcdB对细胞内溶酶体转运和自噬的影响尚不清楚，可能导致更有效地释放到胞浆中，并减少致病葡萄糖转移酶结构域的降解。特异性毒素突变体的产生将有助于研究导致溶酶体功能抑制的分子机制。Galectin-3或Galectin-8在溶酶体上的积聚可能预示着内膜的损伤，并可能为毒素诱导的溶酶体膜完整性的破坏提供线索。药物抑制剂可能有助于揭示钙通道的参与，就像TcdB的早期细胞毒性作用一样。L类钙通道阻滞剂硝苯地平能够消除这种早期的细胞毒性作用。尽管TcdB诱导的溶酶体抑制对硝苯地平不敏感，但不能排除与其他钙通道的类似相互作用。电子显微镜将支持溶酶体和自噬小体的免疫荧光分析，并提供这些细胞器质量和数量改变的证据，以及关于溶酶体功能障碍和自噬小体聚集的原因的信息。通过重组生产和修饰各种梭状芽胞杆菌葡萄糖转移酶和其他毒素，我们能够区分这类毒素的毒素特异性作用和一般作用。所获得的知识有助于更好地了解艰难梭菌相关疾病的发病机制及其频繁复发。

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.