新型胆酸类似物CCD052的抗艰难梭菌作用及其机制研究

艰难梭菌感染是全球重要公共卫生问题，治疗失败和疾病复发常见，现有治疗药物不能完全满足临床需求。新近临床研究提示熊去氧胆酸可治疗复发病例；我们前期研究发现了一类新型的胆酸类似物，其中CCD052体外抗菌活性最强。结合该化合物结构和既往文献推测：CCD052窄谱强效，有独特的胆酸相关的抗菌作用机制，包括特异的作用通路和抑制生长定植的作用靶点。研究拟用人类肠道模型和金黄地鼠生存实验来测定其活性、对肠道菌群和胆酸代谢的影响；用丙酮酸铁氧化还原酶和硝基还原酶活性测试探索其干扰艰难梭菌代谢的机制；用转录组分析、诱导耐药实验和全基因组比较来筛选抑制芽孢萌发、生物膜形成和细菌黏附中胆酸相关关键基因，并用CRISPR基因敲除验证；用免疫印迹法检测NF-κB信号通路被抑制程度；用超高效液相色谱-质谱联合来寻找其它可能的抗菌作用靶标蛋白。研究将系统阐明新型胆酸类似物的抗菌作用机制，为新药研发提供依据。

Clostridioides difficile infection is an important global public health problem. Treatment failure and recurrence of the disease are common. Current therapy cannot fully meet clinical needs. Recent clinical researches indicate that ursodeoxycholic acid may treat recurrent cases. Our previous research found a new class of bile acid analogs, of which CCD052 has the strongest activity in vitro. Based on the literatures and the structure of this compound, our hypothesis is that CCD052 is a narrow-spectrum and highly active antibiotic with unique mechanism related to cholate metabolism, especially specific pathways and targets for inhibiting growth and colonization. We will determine its antibacterial activity against C. difficile and effects on both intestinal flora and bile acid metabolism by human intestinal model and hamster survival trials. We will explore how it interferences the metabolism of C. difficile by pyruvate:ferredoxin oxidoreductase and nitroreductase inhibiting test. Transcriptome analysis, antibacterial resistance inducing experiments, and whole-genome comparisons will be used to reveal key genes related to inhibition of spore germination, biofilm formation and bacterial adhesion, which will be confirmed by CRISPR knockout experiments. Western blot will be used to detect the inhibition of NF-κB signaling pathway. Ultra-performance liquid chromatography-mass spectrometry will be used to reveal potential target proteins related to antibacterial action. This study will explain the antibacterial mechanism of a new type of bile acid analogues, which will provide basic information for development new medicines against Clostridioides difficile.