【摘要】： IPF7817是白念珠菌的功能未知基因,已有研究結(jié)果提示該基因可能參與白念珠菌的耐藥和細(xì)胞內(nèi)氧化還原狀態(tài)的調(diào)節(jié),但是其確切的生物學(xué)功能在本研究開展之前尚不明確。本研究利用基因敲除技術(shù)系統(tǒng)性地考察了IPF7817的功能:首先應(yīng)用“Ura-blaster”策略將IPF7817的兩個等位基因從白念珠菌的基因組中敲除,繼而考察了IPF7817基因敲除對細(xì)胞內(nèi)氧化還原穩(wěn)態(tài)和耐藥表型的影響。氧化還原穩(wěn)態(tài)相關(guān)實(shí)驗(yàn)結(jié)果顯示:IPF7817基因敲除后,細(xì)胞內(nèi)的活性氧水平顯著提高;線粒體功能的直接指標(biāo)——線粒體膜電位升高;一些重要的氧化還原相關(guān)基因(包括GLR1、SOD2和TRR1)轉(zhuǎn)錄表達(dá)上調(diào);細(xì)胞內(nèi)還原型谷胱甘肽和氧化型谷胱甘肽的比值增加。上述結(jié)果提示:IPF7817在白念珠菌的氧化還原穩(wěn)態(tài)調(diào)節(jié)中發(fā)揮重要作用�？疾霫PF7817基因?qū)δ退幈硇陀绊懙膶?shí)驗(yàn)結(jié)果顯示:IPF7817基因的缺失對白念珠菌的藥物敏感性沒有明顯影響;對白念珠菌藥物外排功能沒有明顯影響;但是會導(dǎo)致多藥耐藥基因MDR1的表達(dá)上調(diào);分別用6μg/mL和24μg/mL的氟康唑誘導(dǎo)白念珠菌耐藥性產(chǎn)生,MIC_(80)檢測、Spot assay和Filter disk assay結(jié)果一致顯示:IPF7817基因缺失菌產(chǎn)生氟康唑抗性的時間明顯早于親本菌,并且對其他唑類藥物存在交叉耐藥性;經(jīng)氟康唑誘導(dǎo)后的基因缺失菌JH2_(F6-47)、JH2_(F24-48)相對于誘導(dǎo)后親本RM1000_(F6-47)、RM1000_(F24-48),唑類藥物作用的靶酶基因ERG11表達(dá)顯著上調(diào);敏感性回復(fù)實(shí)驗(yàn)顯示:6μg/mL氟康唑誘導(dǎo)的耐藥親本菌可恢復(fù)對氟康唑的敏感性(MIC_(80)為0.5μg/mL),但同樣條件下誘導(dǎo)的耐藥IPF7817基因缺失菌在40天后仍保持耐藥性的穩(wěn)定,不發(fā)生對氟康唑敏感性的回復(fù)。此外,本研究還考察了IPF7817對白念珠菌菌絲和生物被膜形成能力的影響,IPF7817基因缺失菌和親本菌的結(jié)果未見明顯差異。綜上所述,IPF7817參與了白念珠菌細(xì)胞內(nèi)氧化還原穩(wěn)態(tài)的調(diào)節(jié),并影響白念珠菌誘導(dǎo)耐藥性的產(chǎn)生。
[Abstract]:IPF7817 is an unknown functional gene of candida albicans. The results suggest that the gene may be involved in the regulation of drug resistance and intracellular redox status of candida albicans, but its exact biological function is not clear before the development of this study. In this study, the function of IPF7817 was systematically investigated by gene knockout technique. Firstly, two IPF7817 alleles were knockout from the genome of candida albicans by "Ura-blaster" strategy, and then the effects of IPF7817 knockout on intracellular redox homeostasis and drug resistance phenotype were investigated. The results of redox homeostasis test showed that the level of reactive oxygen species (Ros) in cells increased significantly after knockout of IPF7817 gene, the mitochondrial membrane potential, the transcriptional expression of some important redox related genes (including GLR1,SOD2 and TRR1), and the ratio of reduced Glutathion to oxidized Glutathion increased. These results suggest that IPF7817 plays an important role in the redox homeostasis regulation of candida albicans. The results of investigating the effect of IPF7817 gene on drug resistance phenotype showed that the deletion of IPF7817 gene had no significant effect on the drug sensitivity of candida albicans, but had no significant effect on the drug efflux function of candida albicans, but it would lead to the up-regulation of the expression of multi-drug resistance gene MDR1. Fluconazole (6 渭 g / mL) and 24 渭 g / mL (fluconazole) were used to induce drug resistance of candida albicans. The results of MIC_ (80) detection of, Spot assay and Filter disk assay showed that the time of fluconazole resistance of IPF7817 gene deleted bacteria was significantly earlier than that of parent bacteria, and there was cross resistance to other zole drugs. The expression of gene deletion bacteria JH2_ (F6 鈮，

本文編號：2507827