插入序列共同区(ISCR1)依赖I类整合子定向转移耐药基因分子机制研究

Antimicrobial resistance has become an important problem in the treatment of clinical infections. The emergence of multi-resistant bacteria is mainly due to the horizontal transfer of antibiotic resistance genes. So far ISCR1 is the most powerful mobilization system that can mediate the resistance of almost every class of antibiotics. Its replication and transfer are related to class 1 integron. But the specific mechanism is not clear. In the previous study, we found that the mobilization system mediated by ISCR1 can be present in the form of a linear and a small circular molecule in the same time in the clinical strains. And the transposase encoded by ISCR1 is the only functional protein in the process. Then, we propose a hypothesis: they can recognize the targeting site through transposase by rolling-circle replication, and transfer their adjacent resistance genes from one class 1 integron to another resulting in resistance proliferation and multidrug resistance. In the present study we will elucidate the mechanism of gene transfer, recognition and recombination by ISCR1 in gram-negative bacilli with the methods of inverse PCR, molecular cloning, conjugation, intragastric administration for mice, expression and purification of protein, chromatin Immunoprecipitation and site-directed mutations to explore the key target site and protein during this process. These will be helpful in finding an appropriate way to interrupt the dissemination of antibiotic resistance in molecular level.

细菌耐药已成为临床感染治疗面临的重要难题。多重耐药菌产生最主要是由于耐药基因的水平转移。插入序列共同区（ISCR1）是目前国内外发现的最高效耐药基因定向转运系统，参与几乎所有种类抗菌药物的耐药。它们的复制和转移与I类整合子相关，但具体机制不明确。前期我们研究发现临床菌株中ISCR1介导的耐药系统能够分别以线性形式和自由环状DNA分子形式同时存在，而ISCR1编码的转座酶是转移过程中的唯一功能蛋白，据此我们提出假说：ISCR1通过滚轮复制方式利用转座酶识别定向位点，将其毗邻的耐药基因从一个I类整合子下游转移到其他I类整合子下游，从而导致耐药性的扩散和多重耐药的产生。本项目将通过反向PCR、转化、接合、小鼠灌胃、蛋白表达纯化、染色质免疫共沉淀和基因点突变等技术来阐明临床革兰阴性杆菌中ISCR1依赖I类整合子定向转移耐药基因的分子机制，探索关键靶位和蛋白，从而有助于寻找阻断细菌耐药播散的新策略。

细菌耐药是临床感染面临的严重挑战，多重耐药菌的产生最主要是因为耐药基因的水平转移。其中插入序列共同区（ISCR1）是目前国内外发现的最高效耐药基因定向转运系统，参与几乎所有种类抗菌药物的耐药。本项目从耐药基因blaPER（介导产超广谱β内酰胺酶）入手，通过收集临床相关菌株，PCR检测阳性菌株，测序ISCR1-blaPER结构上下游，接合试验，PFGE，Southern 杂交试验，反向PCR、克隆转化、PCR产物与受体菌共培养等技术来阐明临床革兰阴性杆菌中ISCR1依赖I类整合子定向转移耐药基因（blaPER）的分子机制。结果表明在我国blaPER主要由ISCR1可移动遗传元件介导，IncA/C型广宿主质粒、插入序列IS26、转座子Tn21、Tn1696及MITE能够携带整个复合I类整合子来促进blaPER的进一步传播。在细菌体内证实ISCR1-blaPER-3’CS这个耐药系统既可以以线性形式存在于复合I类整合子中，又可以以自由环状分子形式存在，并且该现象出现于所有临床阳性菌株，ISCR1通过滚轮复制方式利用转座酶将其毗邻的耐药基因从一个I类整合子下游转移到其他I类整合子下游，从而导致耐药性的扩散和多重耐药的产生。它依附于整合子导致细菌的多重耐药，却又具备整合子没有的分子间、分子内的可转移性，可携带耐药基因在不同菌种间、染色体质粒间及不同大小质粒间水平传播。当前，新型抗菌药物的上市和研发缓慢不前，控制耐药性的快速传播是延缓临床上正在使用的抗菌药物的有效途径。