新型家蝇抗菌肽MDAP-2与G-菌细胞膜相关的抗菌机制研究

Studies showed that, antibacterial activity and antibacterial mechanism of antibacterial peptides are closely associated with their structures. A new type of antibacterial peptide MDAP-2 with unique structure was cloned from housefly based on SSH and RACE technology, and prokaryotic expression was conducted. The MDAP-2 antimicrobial peptide gene was not homologous to any genes in GenBank, it had in vitro antibacterial activity against all of the tested Gram-negative bacteria (including sensitive strains and multiple drug-resistant strains) from clinical isolates, inferring theres was unique antibacterial mechanism with the new antibacterial peptide. Preliminary results of antibacterial mechanism research showed that MDAP - 2 can significantly enhance the cell membrane permeability of the Gram-negative bacteria. therefore, the project team members intend to study the secondary structure of MDAP-2 using circular dichroism(CD) technology; Using transmission electron microscopy (sem) technology to study the ultrastructure of Gram-negative bacteria after the action of the MDAP - 2;Using fluorescent probe technique and flow cytometry to study the membrane penetration ability, the membrane depolarization ability, and cell membrane integrity of Gram-negative bacteria after the action of the MDAP - 2,respectively; Using potassium ion sensitive electrode to study the potassium ion microleakage of Gram-negative bacteria caused by MDAP - 2. And then, the cell membrane asociated antibacterial mechanism of MDAP - 2 were studied and confirmed. It will lay a foundation for the development of new type drugs of inhibitting Gram-negative bacteria.

研究表明，抗菌肽的抗菌活性和抗菌机制与抗菌肽的结构密切相关。项目组采用RACE技术从家蝇体内克隆到一个结构独特的新型抗菌肽基因MDAP-2，并进行了原核表达。该基因与Genebank中收录的所有基因序列均无同源性，且其表达产物只对革兰氏阴性菌（敏感株与多重耐药株）具有强抗菌活性，推断其可能存在独特的抗菌机制。抗菌机制的初步研究结果表明，MDAP-2能够显著增强G-菌细胞膜的通透性，因此，项目组拟采用元二色光谱技术对MDAP-2的二级结构进行研究；采用透射电镜技术对MDAP-2作用后的G-菌超微结构进行研究；采用荧光探针技术和流式细胞术分别对MDAP-2作用后的G-菌的膜透化能力、膜去极化能力、细胞膜完整性进行研究；采用钾离子敏感性电极对MDAP-2导致的G-菌细胞钾离子渗漏情况进行研究。进而对MDAP-2作用于G-菌细胞膜相关机制进行研究和确证，为开发新型抗G-菌药物奠定基础。

【背景】：我国主要依赖抗生素来预防和治疗人类和动物的感染性疾病。随着抗生素的广泛应用甚至滥用，多种微生物对常用抗生素产生了耐药性，给疾病防治带来了极大困难。革兰氏阴性菌是当前医院和社区感染中的主要致病菌，其耐药性呈日益上升趋势，严重威胁着人类和动物健康。因此，加快研究用于治疗革兰氏阴性杆菌的新型抗菌药物已成为当务之急。MDAP-2一种结构独特、仅对G-菌有较强抗菌作用的抗菌肽，推测其可能具有独特的抗菌机制。【研究内容】：1.抗菌肽MDAP-2在溶液中的二级机构；2.MDAP-2对对细菌超微结构的影响；3.MDAP-2对G-菌细胞膜通透性的影响；4.MDAP-2对G-菌细胞的膜去极化能力研究；5.MDAP-2对G-菌细胞膜完整性的影响及其导致的菌体细胞内钾离子渗漏情况研究。【重要结果及关键数据】：1.抗菌肽MDAP-2二级结构中以α-螺旋为主，并存在少量β–折叠结构；2.透射电镜结果显示，MDAP-2能使鸡沙门氏菌的细胞膜严重变形、破损，内容物外泄，导致细菌死亡。3.β-半乳糖苷酶检测试验表明MDAP-2能够使细菌细胞内膜的通透性增加，把β-半乳糖苷酶释放到细胞外，分解ONPG，使得反应体系中A405nm的OD值升高；NPN摄入试验结果表明MDAP-2能够使细菌细胞外膜的通透性增加，导致细菌在420nm处的荧光强度明显增加。4.采用DiBAC4(3)膜电位敏感性探针对抗菌肽作用过的鸡沙门菌细胞进行染色，于流式细胞仪上检测其荧光强度变化，其荧光强度明显增加，可达35.9%，可见MDAP-2可使细菌细胞质膜去极化，导致细菌膜内外电势差增加，进而导致细菌死亡。5.MDAP-2可破坏G-细胞膜的完整性导致细菌细胞膜破损，并造成K+的泄露，K+泄露量与随着抗菌肽与细菌作用的时间的延长而增加，并最终稳定在一定的水平。【科学意义】：为开发抗菌肽类新型抗G-菌药物奠定基础。