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，其在被人体细胞摄取后充当导致细胞损伤的强效酶。毒素引起的细胞损伤会导致严重的疾病，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，其由梭菌（C.）很难C.艰难梭菌相关疾病的发病率和严重性正在增加。然而，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，特别是在C。很难因此，开发毒素抑制剂和新的治疗策略提出了重要的未满足的医疗需求。 我们自己的数据表明，人类肽（如防御素）和补体蛋白可能是一个有前途的方法在这种情况下。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活化产物对细菌毒素的作用，重点是C.很难补体级联作为针对危险分子的先天免疫的“主警报和防御系统”发挥作用。然而，它对细菌毒素的特殊作用尚不清楚。在初步数据中，我们可以确定梭菌肠毒素的细胞摄取和作用模式。我们特别发现，中心补体因子（例如C3）和活化产物（例如C3a、C3a（desArg）、C5a）能够显著削弱TcdA、TcdB、CDT和肉毒梭菌C2毒素对人肠上皮细胞的中毒，并且此外，减少细胞中毒素诱导的底物修饰。这些结果表明，生物毒素活性的补体因子的存在下，降低。然而，这种影响的原因至今尚不清楚。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。最初，我们的特点，这些补体蛋白揭示梭菌肠毒素中和作用。该系统平台使用人肠上皮细胞、人肠类器官和乌尔姆大学建立的人肽库。然后，我们将寻找进一步的毒素抑制补体蛋白和肽，并评估是否可以通过补体因子抑制其他毒素（例如白喉，炭疽）。我们希望阐明迄今为止未知的人类补体和细菌毒素之间的相互作用的潜在机制，从而揭示新的药理学策略，有针对性的毒素抑制。

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.