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

许多致病菌产生蛋白质毒素，一旦被人体细胞吸收，就会成为导致细胞损伤的强效酶。毒素引起的细胞损伤可引起严重的疾病，如破伤风、白喉、霍乱或炭疽。特别重要的是危及生命的假膜性结肠炎，它是由艰难梭菌产生的毒素（肠毒素）TcdA、TcdB和CDT引起的。艰难梭菌相关疾病的发病率和严重程度都在增加。然而，目前还没有足够的治疗策略来对抗这些毒素。此外，抗生素治疗相当有限，特别是在艰难梭菌的情况下。因此，开发毒素抑制剂和新的治疗策略是一个重要的未满足的医疗需求。我们自己的数据表明，在这种情况下，人肽（如防御素）和补体蛋白可能是一种很有前途的方法。因此，在拟议的项目中，我们将详细描述中心人类补体因子及其激活产物对细菌毒素的影响，重点是艰难梭菌的肠道毒素。补体级联的功能是先天免疫对危险分子的“主警报和防御系统”。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方式。我们特别发现，中枢补体因子（如C3）和活化产物（如C3a， C3a(desArg), C5a）能够显著损害TcdA， TcdB， CDT和肉毒杆菌c2 -毒素对人肠上皮细胞的中毒，并且减少了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，造成这种影响的原因到目前为止还不清楚。因此，我们将毒素和补体研究专业知识结合起来，协同计划追求破译人类补体因子和相应裂解产物抑制临床相关细菌毒素的分子机制的总体目标。首先，我们描述了那些补体蛋白已经显示出梭菌肠毒素中和作用。该系统平台使用了乌尔姆大学建立的人类肠道上皮细胞、人类肠道类器官和人类肽库。然后，我们将寻找进一步抑制毒素的补体蛋白和肽，并评估补体因子是否可以抑制进一步的毒素（如白喉、炭疽）。我们期望阐明迄今为止人类补体和细菌毒素之间未知的相互作用的潜在机制，从而揭示靶向毒素抑制的新药理学策略。

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.