白假丝酵母ERG11基因突变分析

Analysis of ERG11 gene mutation in Candida albicans

目的探讨白假丝酵母氟康唑作用的靶酶编码基因(ERG11)突变与氟康唑耐药性的关系。方法选择从尿道、阴道、口咽部、呼吸道、血液和前列腺液分离的10株氟康唑耐药白假丝酵母菌和3株敏感菌株,以抽提的白假丝酵母菌基因组DNA为模板,对ERG11基因序列进行PCR扩增,PCR产物直接DNA序列测定,最后应用BLAST和ClustalW软件对测序结果进行比较。结果13株白假丝酵母菌的ERG11基因序列(以已知序列第1个ATG起始密码的A作为第1个记数单位)共有21个位点发生突变,其中17个同义突变位点和4个错义突变位点;在第348bp位点和第383bp位点,耐药菌株和药物敏感株均可发生D116E和K128T变异;在靶酶活性中心血色素结合区和疏水端螺旋区的对应基因片段,耐药株在第1309bp位点可致V437I变化;在第1320bp位点耐药菌株发生碱基突变,形成A、C基因杂合子,有可能导致N440K变化。结论(1)ERG11基因的D116E和K128T位点变异可能与白假丝酵母菌耐药无关;(2)ERG11基因的V437I和N440K位点的改变,可能与耐药形成有关。

Objective To study the relationship between fluconazole （FCZ）-resistance of Candida albicans and the mutation of ERG11 gene encoding FCZ-targeted enzyme. Method Three strains of FCZ-susceptible and 10 FCZ-resistent C. albicans were isolated from the urethra, vagina, oropharynx, respiratory tract, prostate secretion and blood samples. ERG11 gene was amplified by PCR using C.albicans genomic DNA extracts as the templates and the DNA sequences of the PCR products were determined and compared using BLAST and Clustal-W sofiwares. Results The comparison of ERG11 gene sequences identified mutations at 21 sites in 13 strains, including 17 same-sense and 4 missense mutations. Base substitutions at the sites of 348 bp and 383 bp resulting in D116E and K128T conversion may take place in both drug-resistant and drug-susceptible strains. The point mutation at the site of 1309 bp of FCZ-resistant strain may cause V437I change, and the base inversion at 1320 bp may give rise to A/C heterozygosity mutant ofERGI 1 gene, probably resulting in N440K conversion. Conclusion FCZ-resistance of C. albicans may be associated with point mutations of V437I and N440K in ERGI 1 gene, but not with the point mutations of D166E and KI28T.