新型多黏菌素衍生物AL-6抗鲍曼不动杆菌耐药自发突变株的作用机制研究

Acinetabacter baumannii which carbapenem-resistant was posed the greatest threat to human health of 12 deadly antibiotic-resistant bacteria published by the World Health Organization(WHO). Polymyxin or colistin is usually used as a last resort in treating multi-drug resistant(MDR) A.baumanni infections, but the case of therapeutic failure was still high in clinical because of the spontaneous mutations of the bacteria leading to resistance to this drug in vivo. Although considerable progress has been made towards identifying genes associated with resistance, few studies have investigated the mechanisms regulating resistance in A.baumannii. So, in-depth research on polymyxin resistance mechanism and discovery of new polymyxin derivatives to treat met MDR bacterial infections are urgent requirements. . In our previous study, we found that polymyxin derivative AL-6 has better treatment efficacy and lower toxicity compared with the parent compound in vivo. And it has a strong antibacterial activity against the A.baumannii which are polymyxin resistance via spontaneous mutation. Whole Genome Sequencing and genomic comparisons between the polymyxin resistance strains and the parent, there are many novel mutations in the chromosome. A novel mutation in pmrA(I13M) happen in Ab-39 which MIC is 64µg/ml, and also in iclR, shlB_1 and ptk. Little is known about the relationship of the mutations to resistance phenotype. For these, we proposed following two scientific questions: Whether resistance is modulated in the mutations? What is the antibacterial mechanism of derivative AL-6 to polymyxin resistance strains?. Though addressing these scientific questions, it will explain the mechanism of polymyxin resistance in A.baumannii, and will reveal the novel antibacterial mechanism of polymyxin derivatives. There are some important theoretical and practical significance for new polymyxin derivatives designation and clinical treatment option.

鲍曼不动杆菌被列为耐药细菌TOP12之首。多黏菌素作为治疗耐药鲍曼不动杆菌引起感染的“最后一道防线”，其治疗失败率仍居高不下，原因之一与体内感染细菌的自发耐药突变相关。因此，一方面需要对耐药机制进行深入研究，另一方面需要发现更有效的衍生物。. 申请者经前期研究发现，多黏菌素衍生物AL-6在体内比母体化合物具有更好的疗效和更低的毒性；且对多黏菌素自发耐药突变株具有很强的抗菌活性。经基因组测序发现耐药性最高的Ab-39的pmrA内引入了新突变位点（I13M），iclR、shlB_1和ptk三个基因也存在突变。为此，本研究需解答这些突变是如何导致耐药产生？AL-6为何能有效地抑制这些耐药细菌？. 通过以上问题的解答，不仅能解释鲍曼不动杆菌对多黏菌素产生耐药的机制;而且通过对新型衍生物AL-6作用机制的解答，能够发现多黏菌素类抗生素对鲍曼不动杆菌作用的新机制，对设计新的多黏菌素衍生物具有重要的

细菌的耐药性已经成为一个全球性医学难题，尤其是鲍曼不动杆菌的多药耐药性问题。2012年，我国将鲍曼不动杆菌列为目前最重要的“超级细菌”。在2017年WHO公布的最强耐药细菌TOP12中，其列首位。多黏菌素作为50年代发现的“老药”，重新成为临床治疗多药耐药的鲍曼不动杆菌引起严重感染的“最后一道防线”。但是在临床中，多黏菌素耐药菌、适应性耐药及异质性耐药细菌的感染使死亡率高达35-100%。因此，近年来，国内外众多的科学家进行了大量耐药机制研究及多粘菌素结构修饰物研究工作，希望能够获得具有抗菌活性更强、毒性更低的新一代多黏菌素衍生物。前期研究我们获得了自发突变株Ab-39对多黏菌素的耐药性最高并测序确定其在pmrA、iclR、shlB_1和ptk四个基因上存在突变，同时我们筛选到新型多黏菌素衍生物AL-6对自发突变株Ab-39依旧存在更好的抗菌活性。因此，我们对pmrA、iclR、shlB_1和ptk四个基因突变的耐药机制及AL-6体外抗菌活性及机制进行了深入研究。我们利用基因编辑工具还原了Ab-39菌株的突变点，确定了pmrAI13M是Ab-39菌株产生耐药性的主要突变，比较pmrAI13M突变菌株与野生菌株脂质A的结构表明了pmrAI13M突变引起脂质 A发生磷酸乙醇胺修饰的耐药结构基础，并确定另外三个突变中ptkD569N，iclRY49H两个点突变可以协同pmrAI13M点突变增加鲍曼不动杆菌对多黏菌素的耐药性，同时ptkD569N，ShlBR403H，iclRY49H三个点突变都可以协同pmrAI13M点突变增加生物膜形成。同时我们研究了AL-6对其他临床耐药菌株的抗菌活性，并发现其较多黏菌素E具有更好的杀菌效果，对鲍曼不动杆菌的生物膜、持留菌、异质性耐药具有更好的破坏或抑制作用。对于作用机制，我们进行了蛋白组学研究及体外生化机制初探，分别从宏观及作用方式解析了其通过作用后影响鲍曼不动杆菌的氨基酸代谢通路、糖类代谢通路和信号转换通路来干扰细菌正常生理功能及与细菌外膜具有更强的亲电结合能力，从而更强的破坏外膜达到更好的抗菌效果。此研究成果为阐明鲍曼不动杆菌自发突变产生多黏菌素耐药性机理提供新的思路，同时也为AL-6化合物的药物开发奠定基础。