ERG11基因及CDR1基因在阿萨希毛孢子菌唑类耐药中的作用

T.asahii is the most important pathogens of disseminated trichosporosis，its incidence rate increased significantly in recent years and the mortality rate was as high as 80 to 90 percent. Study has found that 20 ~ 30% of clinical strains are resistant to azole antifungal agents and other antifungal drugs which were commonly used.We have isolated the azole resistant strains from patients. However, the resistant mechanism is unclear. Therefore,it is important to carry out its azole drug resistance mechanisms to find new drug targets and reduce the mortality of patients. It has been known that azole resistance is directly related to the drug target enzyme ERG11 gene and drug efflux pumps CDR1 gene in Candida, Cryptococcus, Saccharomyces cerevisiae, Coccidioides immitis. Based on the principle of conservative and homology of gene, we have successfully amplified ERG11 gene in T.asahii ,which is similar to other fungals and preliminary identified the up regulation in drug-resistant strains. On the basis of which, we try to construct ERG11 and CDR1 single gene and double genetic knockout strains and high expression strains. Through in vitro and animal experiments, to observe the change of drug sensitivity after gene knockout and high expression, we should clear the gene functions of azole resistance in T.asahii. which will lay the foundation for the further mechanisms of resistance and new medicine sites.

阿萨希毛孢子菌（T.asahii）是播散性毛孢子菌病最主要致病菌，近年发病率明显上升，死亡率高达80～90%，且20～30%菌株对唑类等抗真菌药物耐药，我们已分离到唑类耐药株。但目前该菌耐药机制不清，因此开展其唑类药物耐药机制研究，对寻找新的药物作用靶点、开发新型抗真菌药，降低患者死亡率均有重要意义。目前在念珠菌、隐球菌、酿酒酵母、粗球孢子菌等真菌中均发现了与唑类耐药最直接相关的药物靶酶基因ERG11基因及主动外排系统CDR1基因。基于基因保守性及同源性原则，我们在T.asahii中成功扩增了与其它真菌相似的ERG11及CDR基因，初步验证其在耐药株中表达上调。拟在此基础上构建ERG11及CDR1单基因与双基因缺失株及高表达株。通过体外和动物实验，观察基因缺失及高表达后菌株对唑类药物敏感性变化，明确上述基因在T.asahii唑类耐药中的作用，为进一步阐明该菌耐药机制及新药靶位设计奠定基础。

本研究通过体外诱导的方法获得了6株稳定的氟康唑耐药菌株，并从体内分离到天然的耐药菌株，检测并发现了在不同浓度氟康唑作用下耐药株ERG11基因mRNA表达量均高于敏感株。扩增筛选到4个CDR1候选基因，验证后发现其非耐药基因。观察并发现纳米银及中药单体丁香酚对该菌的超微结构具有明显的破坏作用，可抑制其生长。研究并发现THP-1单核细胞参与该菌感染的免疫反应过程并发挥抗感染作用。观察并发现了该菌顶体的超微结构，且细胞松弛素D可以抑制顶体的形成。通过比较不同菌落PCR方法快速鉴定，发现玻璃珠法具有较高的敏感性和阳性率。应用三代测序技术对敏感株及耐药株的基因组进行测序发现ERG11基因在耐药株中有多个SNP位点，耐药株中intron1比敏感株长约300 bp。转录组测序进一步证实耐药相关基因主要集中在包括ERG11在内的麦角固醇这条通路上。以上研究结果初步阐明了ERG11基因与阿萨希毛孢子菌的耐药机制有关，主要是通过其表达量的变化以及基因突变发挥其耐药作用。对于进一步研究该菌的耐药机制以及新药靶点的开发具有重要的科学意义和价值。