不同状态下白念珠菌SAP2、MRR2与伊曲康唑耐药的关系  

Relationship Between SAP2 , MRR2 and Itraconazole Resistance in Candida Albicans in Different States

作　　者：解明君 冯文莉[1,2] XIE Mingjun;FENG Wenli(Shanxi Medical University,Taiyuan 030001,China;Department of Dermatology,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学,山西太原030001 [2]山西医科大学第二医院皮肤性病科,山西太原030001

出　　处：《中国皮肤性病学杂志》2024年第10期1074-1083,共10页The Chinese Journal of Dermatovenereology

基　　金：国家自然基金面上项目(82072262)。

摘　　要：目的初步明确在浮游状态和生物膜状态下白念珠菌毒力因子SAP2和转录因子MRR2对伊曲康唑(ITR)耐药的影响。方法对10株伊曲康唑单耐菌株、10株氟康唑(FCA)/ITR/伏立康唑(VRC)全敏感菌株进行SAP2、MRR2基因测序,分析其基因突变对耐药的影响;通过倒置显微镜及激光共聚焦显微镜观察不同时段下白念珠菌生物膜的形成,并通过酶标仪测定其OD值判断生物膜形成能力与耐药的关系。RT-PCR分别对浮游状态及生物膜状态下临床分离白念珠菌进行SAP2及MRR2表达量的检测,研究二者表达水平与伊曲康唑耐药的关系,并对浮游及生物膜状态下MRR2Δ/Δ菌株SAP2表达量进行检测,进一步明确MRR2与SAP2的关系。结果通过基因测序发现,SAP2基因无错义突变,MRR2基因测序出现12个错义突变位点;通过RT-PCR发现在不同状态下,耐药菌株组SAP2[浮游/生物膜:(1.30±0.39)/(4.29±1.76)]和MRR2[浮游/生物膜:(0.95±0.14)/(1.77±0.80)]基因的表达量均高于敏感菌株组[SAP2浮游/生物膜:(0.80±0.48)/(2.59±0.93);MRR2浮游/生物膜:(0.49±0.32)/(0.98±0.64),P<0.05],在生物膜状态下两基因的表达水平SAP2(3.44±1.63)和MRR2(1.19±0.99)相比浮游状态下[SAP2/MRR2:(1.05±0.50)/(0.81±0.62)]均上调(P<0.05),且在浮游状态下,两基因之间有正相关关系(r=0.66,P<0.05),在浮游及生物膜状态下MRR2Δ/Δ菌株SAP2的表达[浮游/生物膜:(0.80±0.06)/(2.15±0.23)]相对于标准菌株ATCC11006[浮游/生物膜:(1.14±0.07)/(2.90±0.10)]均明显下调(P<0.05);通过倒置显微镜及激光共聚焦显微镜观察生物膜形成过程发现,所有菌株均形成了菌丝及孢子相互交错的生物膜,并通过测定其OD值发现,耐药菌株组生物膜形成能力(0.24±0.02)明显高于敏感组(0.15±0.02,P<0.001),MRR2Δ/Δ菌株的生物膜形成能力(0.12±0.12)明显低于标准菌株组(0.16±0.00,P<0.001)。结论伊曲康唑耐药可能与白念珠菌MRR2基因突变有关;在浮游状态和生物�Objective To analyse the effects of Candida albicans virulence factor SAP2 and transcription factor MRR2 on Itraconazole(ITR)resistance in the planktonic and biofilm states.Methods Sequencing of SAP2 and MRR2 genes on 10 ITR monoresistant strains and 10 FCA/ITR/VRC fully susceptible strains to analyse the effect of mutations on drug resistance.The formation of Candida albicans biofilms under different time periods was observed using an inverted microscope and laser confocal microscope.The relationship between biofilm formation ability and drug resistance was assessed by measuring the optical density(OD)value of the biofilm using an enzyme marker.Detection and analysis of SAP2 and MRR2 expression in clinical isolates of Candida albicans in planktonic and biofilm states respectively,by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)technology.We also examined the expression of SAP2 in MRR2Δ/Δstrains in planktonic and biofilm states to further clarify the relationship between MRR2 and SAP2.Results Gene sequencing revealed no missense mutations in the SAP2 gene,and sequencing of the MRR2 gene revealed 12 missense mutation sites.It was found that the expression of SAP2[planktonic/biofilm:(1.30±0.39)/(4.29±1.76)]and MRR2[planktonic/biofilm:(0.95±0.14)/(1.77±0.80)]were higher in resistant strain group than sensitive strain group in different states[SAP2 planktonic/biofilm:(0.80±0.48)/(2.59±0.93);MRR2 planktonic/biofilm:(0.49±0.32)/(0.98±0.64),P<0.05].The expression levels of both SAP2(3.44±1.63)and MRR2(1.19±0.99)were up-regulated in the biofilm state compared to the planktonic state[SAP2/MRR2:(1.05±0.50)/(0.81±0.62)](P<0.05),and there was a positive correlation between the two genes in the planktonic state(r=0.66,P<0.05),the expression of MRR2Δ/Δstrain SAP2 was significantly down-regulated in both planktonic and biofilm states[planktonic/biofilm:(0.80±0.06)/(2.15±0.23)]relative to the standard strain ATCC11006[planktonic/biofilm:(1.14±0.07)/(2.90±0.10)](P<0.05).All strains form

关 键 词：白念珠菌 耐药性 生物膜 SAP2 MRR2 伊曲康唑 

分 类 号：R756.5[医药卫生—皮肤病学与性病学]