染色体介导肺炎克雷伯菌对多黏菌素耐药的新机制研究

The resistance of carbapenem resistant Klebsiella pneumoniae to colistin has put clinical treatment in an awkward situation. Our preliminary study showed one induced colistin resistant CRKP strain only harbored a missense mutation of mgrB gene without functional change, which implied an unknown resistance mechanism. By means of bioinformatics, the mutation site was located on a promoter for downstreamed gene KPHS-33580 which encode a transmembrane protein with positive charge. The result of promoter prediction showed the mutation enhanced the activity of promoter. Therefore, the study hypothesized that the mutation of upstreamed promoter can elevate the expression of KPHS-33580 gene, decrease negative charge of outer membrane, reduce the affinity with colistin, and eventually resulted in colistin resistance. This study aims to prove the function of KPHS-33580 in the forming of colistin resistance by gene knock out/in and other experiments, clarify the mechanism of upstreamed promoter in regulating KPHS-33580 gene by experiments such as promoter probe analysis, and perform epidemiological study to analysis the incidence and epidemiological characteristics of this new mechanism in colistin resistant CRKP clinical strains. Outcomes from this project can provide a new target for development of antimicrobial drugs.

碳青霉烯类耐药肺炎克雷伯菌（CRKP）对多黏菌素耐药使临床治疗陷入困境。本课题组前期发现1株多黏菌素诱导耐药CRKP菌株仅有mgrB基因发生单核苷酸错义突变，基因回补试验初步验证该点突变不影响mgrB基因功能，提示存在未知耐药新机制。生物信息学分析发现此突变位点位于一个可作用于下游基因（KPHS_33580，编码携带正电荷的跨膜蛋白）的启动子上，启动子预测结果提示突变可增强该启动子活性。故本课题组提出假设：KPHS_33580基因上游启动子突变可引起其表达量增高，使细菌外膜携带净负电荷减少，与多黏菌素亲和力降低引起耐药。本课题拟通过基因敲除与回补等试验，明确KPHS_33580参与多黏菌素耐药；通过启动子探针分析等试验确认上游启动子对KPHS_33580蛋白的调控作用；并通过流行病学研究，分析该新耐药机制在多黏菌素耐药肺炎克雷伯菌中的发生率及流行病学特点。研究成果可为药物研发提供新的靶点。

碳青霉烯类耐药肺炎克雷伯菌（CRKP）对多黏菌素耐药使临床治疗陷入困境。本课题主要研究上游启动子调控KPHS_33580基因表达促进CRKP对多黏菌素耐药的分子机制，探索药物研发新靶点。结果发现原始株和诱导株KPHS_33580基因表达水平及上游启动子活性无明显差异，KPHS_33580基因敲除前后菌株对多黏菌素敏感性无改变，提示KPHS_33580基因不参与介导多黏菌素耐药。进一步收集国内多中心临床分离CRKP菌株493株，发现对多黏菌素耐药率为2.2%。通过比较基因组学分析，11株多黏菌素耐药CRKP中有8株检出mgrB基因突变，另外3株分别检出pmrA、pmrB及phoP基因突变。同时，本研究首次报道PmrB新型突变S62R可能介导CRKP对多黏菌素耐药。研究成果揭示了CRKP对多黏菌素耐药机制的复杂性及多元化，作为后续深入研究的基础。通过研究CRKP在多黏菌素E暴露后染色体序列突变，以及突变与多黏菌素耐药之间的关系，发现CRKP在不同浓度的多黏菌素E选择压力下，均会选择出自发耐药突变株，mgrB基因的完全缺失是造成该类自发突变株对多黏菌素E耐药的最主要机制。研究成果为指导临床合理使用多黏菌素、减少耐药发生提供了重要依据。