天然药物功能蛋白质学科组（赖仞）知识库

鲍曼不动杆菌（Acinetobacter baumannii）是一种广泛存在于自然界和医院环境中的革兰氏阴性菌，抗逆性强、营养需求简单，在多种温度及不同酸碱性的条件下均能繁殖生长。该菌可在人体的皮肤、呼吸道、胃肠道以及泌尿系统等部位定植，继而引发肺炎、败血症、尿路感染、腹腔感染、皮肤软组织感染等相关疾病，其对免疫力低下人群感染性和致病性极强，已成为院内感染的重要条件性致病菌。近年来，随着抗生素在临床上的广泛使用，鲍曼不动杆菌耐药菌株的数量呈现爆炸式增长，加之该菌具有极强的获得性耐药和克隆性传播的能力，使得多重耐药、广泛耐药甚至全耐药的鲍曼不动杆菌呈现世界流行趋势。目前临床上针对该菌治疗的可选择药物十分有限，碳氢霉烯类药物耐药鲍曼不动杆菌位列世界卫生组织“新型抗生素研发重点病原体清单”中第一位。对此除了积极检测已有的药物尝试老药新用外，新型抗菌药物的研发已经迫在眉睫。抗菌肽是一类具有抗菌活性的多肽分子，由宿主基因编码，是宿主自身的免疫防御系统中重要的组成部分。天然抗菌肽通常序列较短，一般由10-40个氨基酸残基组成，在水环境中溶解性良好，在极端的温度条件下和强酸强碱环境中能较好的保持结构稳定性和抗菌活性，且具有杀菌作用快、不易产生耐药性的特点，在新型抗菌药物研发方面具有广阔的前景。 我们课题组前期在金环蛇蛇毒中发现了一个抗菌肽cathelicidin-BF，经过十余年的研究，该抗菌肽已获得国家1.1类新药临床批文（CXHL1700235）。在cathelicidin-BF基础上，我们设计改造了分子量更小，更安全，活性更好的抗菌肽，其中，抗菌肽ZY4表现出非常强的抗鲍曼不动杆菌活性。我们通过抑菌圈实验和MIC实验来检测，ZY4对鲍曼不动杆菌标准菌株及耐药菌株的最小抑菌浓度（MIC）为9.375μg/ml，证明多肽ZY4对鲍曼不动杆菌标准菌株及耐药菌株均具有出良好的抗菌活性。能否成为临床治疗候选药物，其安全性和稳定性是极其重要的指标，因此我们用人的血红细胞检测了多肽ZY4的溶血活性以及用人胚肾细胞系HEK293T检测了多肽ZY4的细胞毒性，研究发现这条多肽无溶血活性及细胞毒性且与血浆在37℃共孵育10小时后仍保持良好的抗菌活性，以上结果为将来的药物开发奠定了良好的基础。在杀菌动力学实验中，我们发现多肽 ZY4的杀菌作用是迅速而且彻底的，在1× MIC（9.375μg/ml）浓度时，多肽 ZY4在10 min内就可表现出明显的杀菌作用，在5×MIC和10×MIC 浓度时, ZY4作用1min后就几乎无鲍曼不动杆菌生长，在3 h之内细菌的数量不会再次增加。为了进一步探究多肽ZY4的杀菌机制，我们用扫描电镜观察与多肽ZY4共孵育后鲍曼不动杆菌的形态学变化，发现经过多肽ZY4处理的菌体表面出现了凹陷，推测是由于多肽ZY4作用于菌体表面，造成凹陷和孔洞导致细菌内容物外泄而死亡。 多肽ZY4不仅在体外对鲍曼不动杆菌呈现出良好的抗菌活性，在体内也同样发挥了强劲的杀菌作用。在构建的鲍曼不动杆菌感染的小鼠败血症模型中，我们通过腹腔注射的方式对小鼠进行ZY4的单药治疗以及与多粘菌素E的联合治疗，结果表明多肽ZY4单药治疗以及与多粘菌素E联用治疗均能很好的控制小鼠血液、肺组织、肝组织、脾组织的载菌量以及血浆中炎症因子的水平，病理切片的结果同样显示多肽ZY4的单药治疗和联合治疗可以使得组织病变症状明显改善，且联合用药治疗效果优于单药治疗。 通过上述实验结果我们可以得到结论：多肽ZY4在体内外对鲍曼不动杆菌均具表现出显著的抗菌活性且杀菌作用迅速彻底，同时其具有良好的安全性和稳定性，与多粘菌素E联用治疗后具有协调效应，有望开发成为临床治疗鲍曼不动杆菌感染的候选药物。

 Acinetobacter baumannii is a Gram-negative bacterium widely distributed in nature and hospital environments. It is highly tolerant to adverse, stress and simple nutrient requirements and it can grow under a variety of physiological environments and different acid and alkali conditions. The bacteria can infect the skin, respiratory tract, gastrointestinal tract, and urinary system of the human body, which may lead to pneumonia, sepsis, urinary tract infection, abdominal infection, skin and soft tissue infection and other related diseases, which are opportunistic infections for people with immunocompetent immunity. It is extremely pathological and has become an important conditional pathogen for nosocomial infections in China. In recent years, with the widespread use of antibiotics in clinical practice, the number of A.baumannii resistant strains have exploded, and the bacteria have acquired strong drug resistance and clonal transmission ability, making multi-drug resistance a huge healthcare predicament. The widespread resistance or even total resistance of A.baumannii to conventional antibiotics presents a worldwide trend. At present, there are very limited choices for the treatment of this bacteria in the clinic. The carbapenem-resistant A.baumannii is even ranked first in the World Health Organization's “List of Key Pathogens for New Antibiotic Research and Development”. In addition to actively testing existing drugs, the development of new and alternative antibiotic therapies is paramount Antibacterial peptides are a class of polypeptide molecules with potent antimicrobial activity, which are encoded by the host gene and are an important part of the host's immune defense system. Natural antibacterial peptides usually have a short sequence and generally consist of more than 20 amino acid residues. They have good solubility in an aqueous environment, and can maintain structural stability and antimicrobial activity under extreme physiological conditions and strong acid and alkali environments. They have broad-spectrum antimicrobial activity, quick microbicidal effect, and low propensity to induce resistance. Moreover, they have exhibited broad prospects for the development of novel antimicrobial and therapeutic agents. In our previous study, we discovered an antibacterial peptide cathelicidin-BF from the snake venom. Ten years later, the antibacterial peptide has obtained national of approval as clinical 1.1 new drug (CXHL1700235). Based on cathelicidin-BF, we designed a series of antibacterial peptides with smaller molecular weight and better activity. Among them, antibacterial peptide ZY4 showed very strong activity against A. baumannii. First, the inhibition experiment show that the minimum inhibitory concentration (MIC) of ZY4 against A.baumannii standard strains and resistant strains were both 9.375 μg/ml. The results showed that peptide ZY4 had good antibacterial activity against the standard and resistant strains of A.baumannii. Whether it can become a clinical treatment candidate drug, its safety and stability are extremely important indicators. Therefore, we used human red blood cells to detect the hemolytic activity of the polypeptide ZY4 and the human embryonic kidney cell line HEK293T to detect the cytotoxicity of the polypeptide ZY4. This peptide has low hemolytic activity and cytotoxicity and maintains good antibacterial activity even after incubation with plasma at 37 °C for 10 hours. The above results lay a good foundation for future drug development. In the bactericidal kinetics experiment, we found that the bactericidal effect of peptide ZY4 was rapid and thorough. At 1× MIC concentration（9.375μg/ml）, it showed significant bactericidal effect within 10 min. At 5×MIC and 10×MIC concentration, the growth of A.baumannii was completely inhibited after 1 minute co-incubation with ZY4, there was almost no detectable growth of A.baumannii, and the number of bacteria did not increase again within 3 hours. In order to further explore the bactericidal mechanism of the peptide ZY4, we observed the morphological changes of A.baumannii after co-incubation with the peptide ZY4 by scanning electron microscopy. It was found that the surface of the bacterial cells treated with the peptide ZY4 showed significant disruption and pore formation compared to untreated cells, which was supposedly due to the action of the polypeptide ZY4. Disruption and pore formation on the cell wall of the bacteria resulted in leakage of cellular content and subsequent death. The peptide ZY4 not only exhibits good antibacterial activity against A.baumannii in vitro, but also exerts a strong bactericidal action in vivo. In the constructed sepsis mouse model of A.baumannii infection, we administered the compound ZY4 monotherapy to the mice by intraperitoneal injection and also in combination with colistin. The results showed that the peptide ZY4 monotherapy administration and also in combination with colistin can control the blood volume, lung tissue, liver tissue, spleen tissue and the level of inflammatory factors in plasma. Histological examination results of both single peptide treatment and combined treatment can significantly improve the symptoms of tissue lesions, and the combined treatment exhibited better results than monotherapy administration.Through the above experimental results, we can conclude that the polypeptide ZY4 has significant antibacterial activity against A.baumannii in vitro and in vivo, and the bactericidal effect is rapid and thorough, and it has good safety and stability, and colistin combined therapy has a synergistic effect and is expected to be developed as a candidate for clinical treatment of A. baumannii infection.