中国科学院昆明动物研究所机构知识库

感染性疾病一直以来都是人类难以攻克的难题，其高发病率和高致死率使其在临床和生活中备受关注，细菌感染是最常见的感染性疾病。细菌感染会引起很多疾病，包括尿路、胆道、胃肠道、呼吸道疾病等。细菌感染通常分为胞外菌感染和胞内菌感染两类。单核增生李斯特菌(Listeria monocytogenes，LM)属于胞内菌，是一种人畜共患的致病菌，感染后临床主要表现为败血症、脑膜炎以及孕妇流产，由于其感染率高，危害大，并已成为全球性疾病，因此引起高度重视。近年来随着抗生素的滥用，出现许多耐药细菌，甚至“超级细菌”，使细菌感染的死亡率升高，对人类的健康造成重大的威胁。同时，新型抗生素研发缓慢，因此寻找新型抗感染药物迫在眉睫。 生物活性肽是一种由10-50个氨基酸以不同组成和排列方式构成的复杂的线性或环形结构的不同肽类的总称。其中有毒动物来源的动物活性多肽占据重要的位置。近年来对动物活性多肽研究甚为广泛，研究表明其可以参与机体的代谢和生理调节，其活性包括主要表现有抗菌、抗病毒、抗肿瘤、调节激素、抗糖尿病、降血压、降血脂、调节免疫系统等作用。有研究表明动物活性多肽已经成为重要的抗感染药物开发候选分子，而且被认为在面对耐药性细菌感染时是很有前景的传统抗生素的替代品。基于动物活性多肽具有多样性，高活性，易获得性等特点，使其近年来已逐渐成为创新型药物开发的重要组成部分。实验室前期研究结果显示，眼镜王蛇来源的天然抗菌肽 OH-CATH30 能够利用其直接杀菌能力和选择性的激活天然免疫反应趋化免疫吞噬细胞有效抵御胞外病菌感染。 基于实验室已建立的含有 1300 多条有毒动物来源的天然多肽库，我们初步对部分多肽进行活性筛选，发现一条不具备直接杀菌或抑菌活性而可以清除胞内细菌感染的多肽，即 Rotodorin-F1(CGYRHGRANCGRG)。首先通过直接或间接抑菌实验发现 Rotodorin-F1 不具备直接杀菌或抑菌的活性，随后发现Rotodorin-F1 能显著增强宿主细胞对入侵的单核增生李斯特菌的清除，这些结果提示我们可能与细胞宿主有关，利用宿主自身的免疫能力来清除胞内细菌的感染。实验结果显示 Rotodorin-F1 可以明显减少巨噬细胞（RAW264.7）中单核增生李斯特菌的数量，同时从免疫共定位中得到相同的结果。Western blotting 实验表明Rotodorin-F1 可以增加自噬相关蛋白 LC3-I 向 LC3-II 的转化，P62 的蛋白水平减少，说明 Rotodorin-F1 通过激活自噬达到清除胞内感染细菌的作用。进一步的研究机制表明通过干扰自噬通路相关基因 ATG5，发现阻断自噬通路可以减少Rotodorin-F1 介导的单核增生李斯特菌的数量，揭示了 Rotodorin-F1 可以调节宿主天然免疫来清除胞内细菌感染。小鼠单核增生李斯特菌全身感染实验结果表明Rotodorin-F1 可有效减少感染小鼠体内的单核增生李斯特菌的数量。综上所述，我们的研究表明了 Rotodorin-F1 可以通过自噬相关通路调控宿主细胞天然免疫来抵御胞内细菌的感染，是非常有潜力的治疗胞内细菌感染的潜在药物。更为开发天然活性肽治疗耐药菌的感染提供了更进一步的理解。

Infectious diseases have always been difficult problems for humans to treat.Their high morbidity and high mortality rate have attracted much attention in clinical and life. Bacterial infection is the most common infectious disease. Bacterial infection is an acute systemic infection caused by pathogenic bacteria or conditional pathogens that invade the blood circulation and produce toxins and other metabolites. Bacterialinfections are usually classified into two types: extracellular infections and intracellular infections. Listeria monocytogenes (LM) belongs to intracellular bacteria and. After infection, the main clinical manifestations are sepsis, meningitis and mononucleosis. Due to its high infection rate, It is a hazard and has become a globaldisease, so it is highly valued. The current clinical treatment of Listeria monocytogenes infection mainly includes: antibiotic treatment, bacterial source control, fluid replacement therapy, and regulation of immunity. In recent years, with the abuse of antibiotics, many resistant bacteria and even "super bacteria" have emerged, which has increased the mortality rate of bacterial infections and poses a major threat to human health. At the same time, the development of new antibiotics is slow, so it is extremely urgent to find new anti-infective drugs. A bioactive peptide is a generic term for different peptides ranging from dipeptides to complex linear, circular structures composed of 10-50 amino acids in different compositions and arrangements. Animal active polypeptides derived from toxic animals occupy important positions. In recent years, research on animal active peptides has been extensive. Studies have shown that it can participate in the metabolism and physiological regulation of the body, mainly including antibacterial, antiviral, antitumor, regulating hormones, antidiabetic, lowering blood pressure, lowering blood fat, regulating immune system, etc. effect. Studies have shown thatanimal active peptides have become important candidates for the development of anti-infective drugs, and are considered to be promising alternatives to traditional antibiotics in the face of drug-resistant bacterial infections. Based on the biologica liversity, high activity and easy availability of animal active peptides, it has gradually become an important part of innovative drug discovery in recent years. The results of previous laboratory studies showed that the natural antibacterial peptide OH-CATH30 derived from King Cobra can utilize its direct bactericidal ability to selectively activate the natural immune response to chemotactic immunophagocytic cells to effectively resist extracellular pathogen infection. . Based on the laboratory's established natural peptide library containing more than 1,300 toxic animal sources, we initially screened some of the peptides for activity, and finally found a peptide that does not have direct bactericidal/bacteriostatic activity and can eliminate intracellular bacterial infections, namely Rotodorin. -F1 (CGYRHGRANCGRG). Rotodorin-F1 was first found to have no bactericidal/bacteriostatic activity by direct/indirect bacteriostatic experiments. It was subsequently found that Rotodorin-F1 significantly enhanced host cell clearance of invading Listeria monocytogenes. These results suggest that we may It is related tothe cellular host and utilizes the host's own immunity to remove the infection of intracellular bacteria. The experimental results show that Rotodorin-F1 can significantly eliminate the number of Listeria monocytogenes in macrophages (RAW264.7), and the same results are obtained from immune colocalization. Western blotting showed that Rotodorin-F1 significantly up-regulated LC3II/I and down-regulated the protein level of P62, indicating that Rotodorin-F1 can abolish intracellular infection by activating autophagy. Further research suggests that by blocking the autophagy pathway-related gene ATG5, it was found that blocking the autophagy pathway can eliminate Rotodorin-F1-mediated clearance of Listeria monocytogenes, revealing that Rotodorin-F1 can regulate host natural immunity to clear Intracellular bacterial infection. The results of whole body infection of mouse Listeria monocytogenes showed that Rotodorin-F1 can effectively eliminate bacteriain infected mice and improve the survival rate of mice.. Taken together, our study demonstrates that Rotodorin-F1 is a potential drug for the treatment of intracellular bacterial infections by regulating the innate immunity of host cells through autophagy-related pathways to protect against intracellular bacterial infections. This study not only clarified the molecular mechanism of Rotodorin-F1 scavenging intracellular bacterial infection, but also provided a further understanding of the development of natural active peptides in the treatment of drug-resistant bacteria in immunomodulatory activity.