基于免疫调节发掘有毒动物来源天然多肽抗耐药胞内细菌感染

Intracellular bacterial infection (such as Mycobacterium tuberculosis, Listeria monocytogenes，Salmonella typhimurium) is a serious threat to human health and leads to huge medical cost burden. Currently, the treatment of intracellular bacterial infections mainly relies on the direct antibacterial activity of antibiotics by targeting on bacteria. Intracellular bacterial resistance readily occurs under high stress condition caused by antibiotics, and intracellular bacterial resistance is more and more serious. Thus, novel therapeutic strategies are urgently needed against antibiotic-resistant intracellular bacterial infection. Naturally occurring peptides derived from animals provide a large number of sources for drug development. However, anti-infectious peptides in development mainly rely on their direct bactericidal activity. In this study, we aim to investigate whether modulating the host innate immunity by naturally occurring peptide isolated from toxic animal is a promising therapeutic against antibiotic-resistant intracellular bacterial infection. A peptide library containing more than 1300 naturally occurring peptide have been established in our preliminary work. Intracellular bacteria-infected cell models were employed to screen the peptide library to identify candidate peptides that target on host innate immunity system but not bacteria itself against intracellular bacterial infection. In addition, the efficacy and mechanism of action of candidate peptides will be further investigated in cell model and animal model. Our study may facilitate the further understanding of the action of mechanism of immunomodulatory peptides derived from toxic animal against antibiotic-resistant intracellular bacterial infections, and provide a new strategy in drug development against antibiotic-resistant intracellular bacterial infection.

胞内细菌感染（如结核分枝杆菌、李斯特菌、沙门氏菌等）严重威胁人类健康，造成巨大的医疗负担。针对胞内细菌感染，目前主要是依靠抗生素的直接抑菌或杀菌作用（作用于细菌）。胞内细菌在抗生素的高选择压力下易产生耐药性且耐药问题日趋严峻，迫切需求治疗耐药胞内菌感染的新药物。动物活性多肽为创新型药物研发提供了大量的优秀先导分子，但目前在抗感染药物研发中主要是基于其直接杀菌作用。本研究拟从免疫调节（作用于宿主）的角度出发，基于我们已构建的含有超过1300条来源于有毒动物的多肽库，定向发掘不具有直接抗菌活性，但能通过调节宿主天然免疫抵御耐药胞内细菌感染的天然活性多肽；并利用细胞模型、动物模型深入研究其抵御胞内细菌感染的功能和机制。研究结果有助于深入理解有毒动物来源的活性多肽调节宿主免疫抵御耐药胞内菌感染的功能与机制，为抗耐药胞内细菌感染的创新药物研发提供新策略。

细菌感染，尤其是胞内细菌感染，严重威胁着人类的健康，造成巨大的医疗负担。治疗胞内细菌感染的常规方法是抗生素治疗，由于传统抗生素主要是直接作用于胞内细菌自身，胞内细菌容易对抗生素产生耐药性且耐药情况日趋严峻，更为严重的是近几十年抗胞内细菌感染的抗生素研发已进入瓶颈阶段。有毒动物来源的天然多肽为创新型药物研发提供了大量宝贵的前体分子，但目前在抗感染药物研发中主要是基于其直接杀菌作用。在本项目研究中我们基于免疫调节发掘有毒动物来源的活性多肽抗胞内细菌感染并研究其作用机制。通过单增李斯特菌感染细胞模型筛选了来源于有毒动物的多肽库，发现臭蛙皮肤分泌的活性肽Rotodorin-F1（CGYRHGRANCGRG）不具有直接的抑菌、杀菌活性，但是能显著增强宿主细胞（上皮细胞、巨噬细胞）抵御单增李斯特菌、沙门氏菌及海洋分枝杆菌侵染的能力。多肽Rotodorin-F1能够调控细胞自我防御能力，尤其是对自噬的调节：呈剂量效应地增强小鼠巨噬细胞以及人肺上皮细胞的自噬能力从而清除胞内感染的细菌。自噬抑制剂、干扰自噬相关基因ATG5表达均能抑制Rotodorin-F1清除胞内感染的细菌能力。通过构效关系研究发现Rotodorin-F1序列中的半胱氨酸和精氨酸对其通过促进宿主细胞自噬抗胞内细菌感染的能力是非常重要的。体外细胞毒性实验及体内动物毒性试验发现Rotodorin-F1在发挥其显著调节自噬抗胞内细菌感染的有效浓度、剂量下并未检测出明显的细胞毒性及对小鼠的急性毒性。在胞内细菌感染的动物模型中，Rotodorin-F1能够显著降低肝、脾中的细菌载量并且提高被感染小鼠的存活率。此外，我们同时用有毒动物大蹼铃蟾皮肤分泌的aerolysin样毒素蛋白βγ-CAT作为多肽Rotodorin-F1调控宿主细胞免疫活性的对比研究，揭示了βγ-CAT通过作用于细胞膜上的酸性糖鞘脂内化进入宿主细胞，在宿主细胞内体、溶酶体上形成孔道调控宿主细胞内体-溶酶体途径促进适应性免疫抗感染的机制。通过本项目的研究表明基于免疫调节发掘抗耐药胞内细菌感染药物是可行的，为抗耐药胞内细菌感染的药物开发及治疗提供了新思路。