摘要
恩波吡维胺(pyrviniumpamoate,PP)是一种传统抗寄生虫药,与唑类联合具有协同抗烟曲霉作用。af-fadO(FADbindingoxidoreductase,FAD依赖性氧化还原酶)基因的编码产物在烟曲霉中推定为一种与FAD结合的氧化还原酶,可能参与真菌的氧化呼吸过程。通过敲除烟曲霉af-fadO基因,构建烟曲霉af-fadO基因的敲除株,揭示该基因及其编码蛋白在PP与唑类协同抗烟曲霉中的作用,并探索了其对烟曲霉药物敏感性和渗透压、氧化压力物质敏感性的影响。本研究采用同源重组构建出af-fadO基因的敲除盒,筛选出符合的烟曲霉af-fadO敲除株Δaf-fadO。利用纸片法和棋盘法观察该突变株与野生株WT(ku80,pyrG^(+))在PP与唑类(泊沙康唑、伊曲康唑、伏立康唑)联合抗烟曲霉中的敏感性变化。同时,观察该突变株与野生株WT对氧化压力(H_(2)O_(2)和甲萘醌)、渗透压物质(NaCl、D-山梨醇)敏感性的变化。体外联合药物敏感性试验结果显示Δaf-fadO对PP与泊沙康唑的分级抑菌浓度指数(fractional inhibitory concentration index,FICI)为0.563,显示无相互作用,而对照组WT对PP与泊沙康唑的FICI值为0.5,存在协同作用。纸片法结果发现PP与泊沙康唑联合对Δaf-fadO的抑菌圈比对照组WT产生的抑菌圈直径明显变小,PP与泊沙康唑联合时对敲除株抑制作用更显著,差异有统计学意义(P<0.05)。在渗透压和氧化应激的培养试验中,与野生株WT相比较,Δaf-fadO对氧化剂H_(2)O_(2)、甲萘醌、NaCl和D-山梨醇更敏感。敲除烟曲霉af-fadO基因后,其对氧化和渗透压力的敏感性增加,逆转了PP与泊沙康唑的协同作用,af-fadO可能是PP作用于烟曲霉的靶点。本研究丰富了PP协同唑类抗真菌的机制研究,为将来的临床应用奠定了基础。
Pyrvinium pamoate(PP)is an antiparasitic drug,which has been found to exert synergistic effect with azoles against Aspergillus fumigatus.The putative protein encoded by the af-fadO gene in A.fumigatus is a FAD binding oxidoreductase,which might be involved in the respiration chain in the mitochondria.af-fadO knockout strain was constructed to investigate the role of the gene in antifungal susceptibility,osmotic and oxidative stress adaption of A.fumigatus,and the synergistic effect of PP and azoles.af-fadO knockout strain was constructed via homologous gene replacement method.The effect of af-fadO disruption on the in vitro antifungal susceptibility,oxidative and osmotic stress adaption as well as the effect on the combinational interactions between PP and azoles(posaconazole,itraconazole,voriconazole)in the mutant Δaf-fadO and wild-type(WT)(ku80,pyrG^(+))strains was observed.The effect of the gene on synergism of PP and azoles was investigated via broth microdilution checkerboard technique and disk diffusion method.In vitro antifungal susceptibility assay revealed that fractional inhibitory concentration index(FICI)value of PP combined with posaconazole against Δaf-fadO was 0.563,indicating no interaction,whereas the FICI against WT was 0.5,indicating synergistic effect.The results of disk diffusion method showed that the inhibitory zone diameter of PP combined with posaconazole against Δaf-fadO was significantly smaller as compared with that against WT.In addition,Δaf-fadO was more sensitive to the oxidants H_(2)O_(2) and menaphthoquinone,osmotic agents NaCl and D-sorbitol as compared with WT.The disruption of af-fadO gene in A.fumigatus results in mutantΔaf-fadO showing more sensitivity to oxidative and osmotic stress,reversing the synergistic effect of PP and posaconazole.The present study enhances the knowledge of PP and azole antifungal mechanism,and is valuable for future clinical application.
作者
程文旭
沈敏
张恒
龚霄
孙毅
高露娟
万俐佳
CHENG Wenxu;SHEN Min;ZHANG Heng;GONG Xiao;SUN Yi;GAO Lujuan;WAN Lijia(Department of Otolaryngology,Jingzhou Hospital Affiliated to Yangtze University,Jingzhou 434020,Hubei,China;Department of Dermatology,Jingzhou Hospital Affiliated to Yangtze University,Jingzhou 434020,Hubei,China;Department of Dermatology,Zhongshan Hospital(Xiamen),Fudan University,Xiamen 361015,Fujian,China;Department of Dermatology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《菌物学报》
CAS
CSCD
北大核心
2024年第2期38-46,共9页
Mycosystema
基金
福建省自然科学基金面上项目(2021D031)
医疗卫生指导性项目(厦门市科学技术局3502Z202114ZD1072)
湖北省重点研发项目(大健康领域,2022BCE022)
湖北省卫生健康委科技项目(WJ2021M261)
荆州市科技局科技项目(2022HC61)。
关键词
烟曲霉
FAD依赖性氧化还原酶
恩波吡维铵
泊沙康唑
Aspergillus fumigatus
FAD binding oxidoreductase(af-fadO)
pyrvinium pamoate(PP)
posaconazole
作者简介
Corresponding authors:孙毅,E-mail:jzzxyysy@163.com,ORCID:0000-0002-4489-3803;Corresponding authors:高露娟,E-mail:gao_lujuan@163.com;程文旭,ORCID:0009-0005-1010-4676。
