法尼醇对白念珠菌生物膜耐药相关信号通路的调控机制

Biofilms are complex architectures of different cell types encompassed within an extracellular matrix and are extremely resistant to most antifungals. C. albicans biofilms resistance poses a critical challenge to clinical therapy. Studies show that singaling cascades are the foundation of the biofilms formation, and a correlation exists between biofilm formation and morphogenesis, and their resistance. Our previous studies have proven that quorum sensing molecules such as farnesol and tyrosol regulate morphogenesis and biofilms formation, and that farnesol can decrease fluconazole resistance by regulating the sterol biosynthetic pathway genes ERG. It has been proven that cAMP-dependent PKA and MAPK singaling cascade is of central importance for C. albicans morphogenesis, quorum sensing molecules such as farnesol regulate the yeast-hyphal transition by cAMP-dependent PKA and MAPK cascade. The cAMP-dependent PKA and MAPK also play a signicicant role in the regulation of C. albicans resistance. Studies showed that C. albicans regulate the sterol biosynthesis by activating cAMP-dependent PKA and MAPK singaling cascades. In this study we hypothesize that farnesol or change C. albicans membrane morpgologensis by regulating genes and proteins expression in the cAMP-dependent PKA and MAPK singaling cascade, which leads to the decreasing of biofilms resistance. To test this hypothesis, the expression of the related genes and proteins in cAMP-PKA and MAPK singaling cascade will be analysed by quantitative PCR(qPCR) and western blot test respectively. Meanwhile, we are to measure the MICs to compare drug resistances by using a formazan salt reduction assay(XTT assay), and to observe the morphology changes by using Confocal laser scanning microscopy (CLSM) and Scanning electron microscope (SEM) on quorum sensing various treated and untreated and fluconazole-resistant groups. The cell membrane sterols will be analysed by chromatography and mass spectrometry. Furthermore, effects of farnesol on the mice model of systemic candidiasis or oral candidiasis are carried on to learn more about its clinical evaluation in the future. This study will help us to better understand the C. albicans biofilms resistance mechanism, which is very important in identifying new targets for antifungal drugs.

白念珠菌生物膜耐药是临床亟待解决的关键问题之一。白念珠菌耐药与细胞形态转化和生物膜形成密切相关。我们前期研究证实法尼醇抑制白念珠菌酵母相向菌丝形态转化和生物膜形成，并通过调控麦角甾醇合成通路基因表达而增加药物易感性。已知cAMP-PKA和MAPK信号通路既是法尼醇调控信号通路，又是白念珠菌耐药相关信号通路。法尼醇通过cAMP-PKA和MAPK信号通路调控菌丝形态变化，白念珠菌通过激活cAMP-PKA和MAPK信号通路参与麦角甾醇耐药。本研究假设法尼醇通过cAMP-PKA和MAPK信号通路基因和蛋白表达而调控细胞质膜形态转化，从而改变生物膜药物易感性。本课题采用实时定量PCR、免疫印迹和高效液相色谱分别检测基因、蛋白表达及细胞质膜甾醇水平，药物易感性分析检测耐药性，激光共聚焦和扫描电镜观察形态学改变，体外及动物实验进行疗效评估。本研究有助于阐明生物膜耐药机制，为寻找抗真菌药物靶标提供理论依据

本研究发现法尼醇改变白念珠菌生物膜MAPK和cAMP-PKA信号通路基因和蛋白的表达，其中RAS1、CYR1和PDE2是主要调控基因。法尼醇抑制（6 h、12 h、24 h和 36 h）RAS1基因和蛋白表达。法尼醇抑制CYR1基因和蛋白表达（6 h、24 h和36 h），增高PDE2基因和蛋白表达（6 h、12 h和36 h）。构建白念珠菌RAS1基因高表达和基因敲除株，药敏实验显示RAS1基因与浮游和生物膜状态白念珠菌的耐药相关。RAS1基因高表达可以提高白念珠菌生物膜耐药性，RAS1基因敲除株可降低生物膜耐药性。法尼醇处理RAS1基因高表达组显著降低其对抗真菌药物耐药性，而法尼醇处理RAS1基因敲除株组未见耐药性显著性变化。进而，法尼醇对RAS1基因高表达株中RAS1基因和蛋白表达起负向调控作用。激光共聚焦显微镜显示法尼醇可以抑制RAS1基因高表达株生物膜形成；此外，RAS1基因高表达株生物膜形成能力高于RAS1基因敲除株。构建CYR1和PDE2高表达株，药敏实验显示CYR1和 PDE2与浮游状态和生物膜白念珠菌耐药相关。CYR1和PDE2高表达株增高生物膜对两性霉素B的耐药性。法尼醇可以降低CYR1和 PDE2高表达株生物膜对抗真菌药物的耐药性。进而，法尼醇对白念珠菌 CYR1基因高表达株生物膜的CYR1基因及蛋白起负向调控的作用，而对PDE2基因高表达株的PDE2基因及蛋白起正向调控作用。激光共聚焦显微镜发现，法尼醇可明显抑制 CYR1和PDE2基因高表达株的生物膜形成；此外，CYR1基因高表达株、PDE2基因高表达株生物膜形成能力高于野生对照株，PDE2基因高表达株生物膜形成能力高于CYR1高表达株。采用FICI法和ΔE法对法尼醇与抗真菌药物联合对白念珠菌生物膜作用的体外研究发现，法尼醇与5-氟胞嘧啶、法尼醇与氟康唑联合有协同效应；法尼醇与特比萘芬联合、法尼醇与伊曲康唑联合有拮抗效应。法尼醇与抗真菌药物的相互作用在生物膜的不同时相中存在差异。随后，构建白念珠菌口腔黏膜感染小鼠模型，法尼醇（50 μM）与制菌霉素（50 μ g/ml）联合、2个浓度法尼醇（4 μM、8 μM）分别与伊曲康唑联合存在协同作用，而法尼醇单独使用对白念珠菌口腔黏膜感染小鼠无作用。