Inhibition of bacterial protein toxins, in particular the toxins of Clostridioides difficile, by factors and cleavage products of the human complement system

许多致病细菌产生蛋白质毒素，这些毒素一旦被人类细胞吸收，就会作为强有力的酶而导致细胞损伤。毒素诱导的细胞损伤会导致破伤风、白喉、霍乱或炭疽等严重疾病。尤其重要的是危及生命的伪膜性结肠炎，它是由Clostridioides(C.)产生的毒素(肠毒素)TcdA、TcdB和CDT引起的。艰难抉择。艰难梭菌相关疾病的发病率和严重性正在增加。然而，目前还没有针对这些毒素的足够的治疗策略。此外，抗生素治疗相当有限，特别是艰难梭菌。因此，开发毒素抑制剂和新的治疗策略提出了一个重要的未得到满足的医学需求。我们自己的数据表明，人源多肽(如防御素)和补体蛋白在这方面可能是一种有前途的方法。因此，在拟议的项目中，我们将详细描述中央人类补体因子及其激活产物对细菌毒素的影响，重点放在艰难梭菌的肠毒素上。补体级联起到了针对危险分子的先天免疫的“主警报和防御系统”的作用。然而，它对细菌毒素的特殊作用尚不清楚。在初步数据中，我们可以确定梭状芽胞杆菌肠毒素的细胞摄取和作用模式。我们特别发现，中枢补体因子(如C3)和激活产物(如C3a、C3a(DesArg)、C5a)能够显著减轻TcdA、TcdB、CDT和肉毒梭菌C2-毒素对人肠上皮细胞的毒性，并减少毒素诱导的细胞底物修饰。这些结果表明，补体因子的存在降低了生物毒素的活性。然而，造成这种效果的原因到目前为止还不清楚。因此，我们结合了我们的毒素和补体研究专业知识，并协同计划，以追求破译人类补体因子和相应的裂解产物抑制临床相关细菌毒素的分子机制的首要目标。首先，我们对那些显示出梭状芽胞杆菌肠道毒素中和作用的补体蛋白进行了表征。该方法平台使用了乌尔姆大学建立的人类肠道上皮细胞、人类肠道器官和人类多肽库。然后，我们将寻找更多抑制毒素的补体蛋白和多肽，并评估补体因子是否可以抑制更多的毒素(如白喉、炭疽)。我们希望阐明迄今为止未知的补体和细菌毒素之间相互作用的潜在机制，从而揭示靶向毒素抑制的新的药理策略。

Many pathogenic bacteria produce protein toxins, which upon uptake by human cells act as potent enzymes resulting in cellular damage. The toxin-induced cell injury causes severe diseases such as tetanus, diphtheria, cholera or anthrax. Of particular importance is the life-threatening pseudomembraneous colitis, which is caused by toxins (enterotoxins) TcdA, TcdB and CDT, which are produced by Clostridioides (C.) difficile. C. difficile associated diseases are increasing in incidence and severity. However, there are no sufficient therapeutic strategies against these toxins. Furthermore, antibiotic treatment is rather limited, especially in the case of C. difficile. Therefore, development of toxin inhibitors and novel therapeutic strategies poses an important unmet medical need. Our own data indicate that human peptides (e.g. defensins) and complement proteins may represent a promising approach in that context. Thus, in the proposed project, we will characterize in detail the effects of central human complement factors and their activation products in regard to their effects on bacterial toxins with a focus set on enterotoxins from C. difficile. The complement cascade functions as a “master alarm and defence system” of innate immunity against danger molecules. However, its particular role against bacterial toxins is unknown. In preliminary data, we could define the cellular uptake and mode of action of clostridial enterotoxins. We especially found that central complement factors (e.g. C3) and activation products (e.g. C3a, C3a(desArg), C5a) were capable to significantly impair intoxication of human intestinal epithelial cells by TcdA, TcdB, CDT, and Clostridium botulinum C2-Toxin, and furthermore, reduced toxin-induced substrate modification in the cells. These findings suggest that the biological toxin activity was decreased in presence of complement factors. However, the reason for this effect is not known so far. Thus, we have combined our toxin and complement research expertise and synergistically plan to pursue the overarching aim of deciphering the molecular mechanisms by which human complement factors and corresponding cleavage products inhibit clinically relevant bacterial toxins. Initially, we characterize those complement proteins which have revealed clostridial enterotoxin-neutralizing effects. The methodical platform uses human intestinal epithelial cells, human intestinal organoids, and human peptide banks established at Ulm University. We will then search for further toxin-inhibiting complement proteins and peptides and evaluate whether further toxins (e.g. diphtheria, anthrax) can be inhibited by complement factors. We expect to elucidate the underlying mechanisms of the so far unknown interactions between human complement and bacterial toxins and thereby reveal novel pharmacological strategies for targeted toxin inhibition.