Cellular uptake of the Rho-activating toxins CNF1 and CNFY and of further deamidases

多年来，我的团队一直在研究细菌毒素（脱酰胺酶），它们分别组成性地激活Rho gtpase或g α-亚基异三聚体g蛋白。我们分析了大肠杆菌、假结核耶尔森菌（CNFY）和肠沙门氏菌（CNFS）分别产生的CNF家族5个成员（细胞毒性坏死性因子1、2、3、CNFY和CNFS）的分子机制。此外，我们还研究了多杀巴氏杆菌毒素（PMT）和博德氏杆菌皮肤坏死毒素（DNT）。除了分子机制外，了解细胞特异性（细胞受体）和进入哺乳动物细胞的摄取途径对于强大的分子工具的细胞生物学使用和药理抑制至关重要。为了鉴定细胞蛋白受体，生物化学方法已被用于纯化高亲和膜蛋白。事实上，一些受体如CNF1结合Lu/BCAM（路德/基底细胞粘附分子）已经被确定。此外，我们还将蛋白聚糖描述为毒素的着陆平台。为了更有效和直接的受体鉴定策略，我们建立了一种基于慢病毒敲除文库的新方法。因此，我们产生了两种稳定的表达Cas9的小鼠细胞系和一种允许每个细胞敲除统计上一个基因的方案。使用嵌合毒素PMT-DTA（白喉毒素的催化结构域），我们已经进行了第一次筛选，并确定了以下基因，这些基因似乎是嵌合毒素结合、摄取和作用所必需的：两个合成双苯二胺的基因（DTA的靶氨基酸，显示出检测的功能），低密度脂蛋白受体相关蛋白1 （LRP1）和寡糖转移酶复合物（Ostc）的亚基。在第一个实验中，lrp1敲除细胞显示出对PMT-DTA的抗性。我们计划研究LRP-1和Ostc与PMT结合和摄取的相关性，并确定确切的相互作用氨基酸。此外，我们希望利用我们的遗传筛选来鉴定进一步的毒素受体相互作用伙伴，特别是耶尔森菌CNFY。

For many years my group studied bacterial toxins (deamidases), which constitutively activate Rho GTPases or the Gα-subunit of heterotrimeric G-proteins, respectively. We analyzed the molecular mechanisms of 5 members of the CNF family (Cytotoxic Necrotizing Factors 1, 2, 3, CNFY and CNFS) produced by Escherichia coli, Yersinia pseudotuberculosis (CNFY) and Salmonella enterica (CNFS), respectively. Moreover, we studied Pasteurella multocida toxin (PMT) and Bordetella Dermo-Necrotzing toxin (DNT). Besides the molecular mechanism, the knowledge about cell specificity (cellular receptor) and uptake route into mammalian cells is essential for the cell-biological use and pharmacological inhibition of the powerful molecular tools.For the identification of cellular protein receptors, biochemical methods have been used to purify high affinity membrane proteins. Indeed, some receptors like the CNF1 binding Lu/BCAM (Lutheran/ Basal Cell Adhesion Molecule) have been identified. Moreover, we characterized proteoglycans as landing platform for the toxins. For more effective and straight forward strategy for receptor identification, we established a new method based on a lentiviral knockout library. Therefore, we generated two stable Cas9 expressing mouse cell lines and a protocol in which allows knockout of statistically one gene per cell. Using a chimeric toxin PMT-DTA (catalytic domain of diphtheria toxin), we already performed the first screens and identified the following genes which seem to be necessary for binding, uptake and action of the chimeric toxin: two genes for diphthamide synthesis (target amino acid for DTA, showing functionality of out assay), LDL Receptor Related Protein 1 (LRP1) and a subunit of the Oligosaccharyltransferase Complex (Ostc). In first experiments, LRP1-knockout cells showed resistance towards PMT-DTA. We plan to study the relevance of LRP-1 and Ostc for binding and uptake of PMT and to define the exact interacting amino acids. Moreover, we want to use our genetic screen for identification of further toxin-receptor interaction partners, especially for Yersinia CNFY.