摘要
目的探讨游离状态和生物膜状态下,毒力因子SAP2、转录因子STP1在临床分离白念珠菌伊曲康唑(ITR)耐药中的作用及其相互关系。方法实验菌株为临床分离伊曲康唑耐药白念珠菌菌株和敏感菌株各10株。首先,采用PCR方法对游离状态下临床分离白念珠菌伊曲康唑耐药和敏感菌株的SAP2、STP1进行测序,分析基因突变与耐药的关系;之后,利用96孔板构建白念珠菌生物膜;采用RT-PCR方法检测游离状态和生物膜状态下白念珠菌SAP2、STP1、STP1Δ/Δ的mRNA表达水平,比较分析不同状态下SAP2、STP1、STP1Δ/Δ的表达是否具有差异和相关性;进一步通过结晶紫染色法和激光共聚焦显微镜观察白念珠菌生物膜在各时间点的生长形态以及SAP2、STP1、STP1Δ/Δ对其形成能力的影响。结果SAP2测序显示无错义突变,存在3个同义突变(T276A、G543A和A675C);STP1基因测序发现两个错义突变N394S和D71I,以及2个同义突变C453T、A799G,其中N394S在全部耐药和敏感菌株中都存在,而D71I仅存在于两株敏感菌株中。与敏感菌株相比,白念珠菌耐药菌株和STP1基因缺陷菌株(STP1Δ/Δ)的生物膜形成能力更强;耐药菌株在游离状态和生物膜状态下SAP2和STP1的表达均上调(P<0.05),并且与游离状态相比,耐药菌株和敏感菌株在生物膜状态下二者的表达量均显著升高(P<0.05)。而无论在何种状态下,SAP2和STP1的mRNA表达水平均呈显著正相关。此外,与标准菌株ATCC11006相比,STP1基因缺陷菌株(STP1Δ/Δ)中的SAP2表达在两种状态下也显著上调,差异具有统计学意义(P<0.05)。结论生物膜的形成可以使白念珠菌伊曲康唑耐药性增加,但SAP2、STP1基因突变可能与其耐药性无关;游离状态下,SAP2的高表达与白念珠菌ITR的耐药性有关,而无论在游离状态还是生物膜状态下,白念珠菌对ITR的耐药性增加均与STP1有关。此外,STP1对SAP2是具有调控作用的,其机制亟待研究。
Objective To investigate the role and relationship of virulence factor SAP2 and transcription factor STP1 in itraconazole(ITR)resistance of Candida albicans in free state and biofilm state.Methods The experimental strains were 10 itraconazole-resistant Candida albicans and 10 sensitive strains.Firstly,the SAP2 and STP1 of itraconazole-resistant and sensitive strains of Candida albicans isolated in free state were sequenced by PCR method,and the relationship between gene mutation and drug resistance was analyzed.Then,the biofilm of Candida albicans was constructed by 96-well plate.The mRNA expression levels of SAP2,STP1 and STP1Δ/Δof Candida albicans in free state and biofilm state were detected by RT-PCR method,and the differences and correlation of SAP2,STP1 and STP1Δ/Δexpression in different states were compared and analyzed.The growth morphology of Candida albicans biofilm at different time points and the effects of SAP2,STP1 and STP1Δ/Δon its formation ability were observed by crystal violet staining and laser confocal microscope.Results SAP2 sequencing showed that there were no missense mutations with three synonymous mutations(T276A,G543A and A675C),two missense mutations N394S(20)and D71I(2)and two synonymous mutations C453T(4)and A799G(1)were found in STP1 gene sequencing,among which N394S existed in all drug resistant and sensitive strains,while D71I only existed in two sensitive strains.Compared with sensitive strains,Candida albicans resistant strains and STP1 gene deficient strains(STP1Δ/Δ)had stronger biofilm forming ability,and the expressions of SAP2 and STP1 were up-regulated in free state and biofilm state,and the expression levels of SAP2 and STP1 in biofilm state were significantly higher in resistant and sensitive strains than in free state.However,there was a significant positive correlation between the mRNA expression levels of SAP2 and STP1 under any condition.In addition,compared with the standard strain ATCC11006,the expression of SAP2 in the STP1 gene deficient strain(STP1Δ/Δ)was also significantly up-regulated under the two states,and the difference was statistically significant.Conclusion The formation of biofilm can increase the itraconazole resistance of Candida albicans,but the mutations of SAP2 and STP1 genes may not be related to the drug resistance of Candida albicans.In free state,the high expression of SAP2 is related to the drug resistance of Candida albicans ITR,while in both free state and biofilm state,the increase of ITR resistance of Candida albicans is related to STP1.In addition,STP1 can regulate and control SAP2,and its mechanism needs to be studied urgently.
作者
虞岱瑶
冯文莉
Yu Daiyao;Feng Wenli(Department of Dermatology and Venereology,Second Clinical Medical College,Shanxi Medical University,Taiyuan 030009,China)
出处
《中华临床医师杂志（电子版）》
CAS
2024年第3期283-294,共12页
Chinese Journal of Clinicians(Electronic Edition)
基金
国家自然基金面上项目(82072262)。
