密度感应分子对白念珠菌生物膜耐药性的作用及其信号通路调控

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. C. albicans regulate the sterol biosynthesis by activating cAMP-dependent PKA and MAPK singaling cascade. In this study we hypothesize that quorum sensing molecules such as farnesol or tyrosol change C. albicans biofilms resistance by regulating genes and proteins expression in the cAMP-dependent PKA and MAPK singaling cascade. To test this hypothesis, 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 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. The cell membrane sterols will be analysed by chromatography and mass spectrometry. 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。研究证实细胞间信号转导是生物膜形成基础，细胞形态转化和生物膜形成与白念珠菌耐药密切相关。我们前期研究证实密度感应分子调控白念珠菌形态转化和生物膜形成，并通过调控麦角甾醇合成通路ERG基因表达而改变药物易感性。已知cAMP-PKA和MAPK信号通路既是密度感应分子调控信号通路，又是白念珠菌耐药相关信号通路。白念珠菌通过激活cAMP-PKA和MAPK参与麦角甾醇耐药，密度感应分子通过这两个信号通路调控细胞质膜形态变化。本研究假设密度感应分子通过调控cAMP-PKA和MAPK信号通路基因和蛋白表达而改变白念珠菌生物膜药物易感性。本课题采用药物易感实验、XTT检测耐药性，激光共聚焦和扫描电镜观察形态，实时定量PCR、免疫印迹和气相色谱-质谱分别检测基因、蛋白表达及细胞质膜甾醇水平。本研究将丰富白念珠菌生物膜耐药机制理论，为寻找抗真菌药物靶标提供理论依据。

白念珠菌是人类口腔常驻条件致病真菌，生物膜是其自然存在方式，白念珠菌生物膜耐药性明显增强。白念珠菌生物膜耐药是临床亟待解决的关键问题。前期研究已经证实密度感应分子调控白念珠菌形态转化和生物膜形成，并调控麦角甾醇合成通路多个ERG基因表达。本申请课题以此为基础，立论于法尼醇对白念珠菌生物膜耐药性作用及其分子机制的研究。由于本项目被批准为面上小额资助，四年完成的计划将要修改为一年完成的研究。因此，本研究内容调整为法尼醇对白念珠菌生物膜耐药性的作用，这一部分是原来研究计划的前提和基础，也是深入进行分子机制研究的技术支持和理论储备。本研究采用下述方法检测法尼醇对生物膜的耐药性：XTT分析SMIC检测药物易感性，激光共聚焦和扫描电镜观察形态，高效液相色谱检测生物膜麦角甾醇含量。.本研究采用氟康唑诱导耐药株，KONT真菌显色MIC药敏检测试剂盒鉴定耐药株。药物易感实验发现法尼醇增高白念珠菌生物膜氟康唑药物易感性。法尼醇抑制白念珠菌生物膜的生长，与未处理对照比较存在显著性差异（p<0.001）。法尼醇对早期生物膜抑菌作用高于成熟期生物膜，差异具有显著性（p<0.05）。法尼醇对白念珠菌易感株和耐药株生物膜的抑菌作用随浓度增高（50μM、100μM、150μM、200μM、250μM和300μM）而逐渐增高；法尼醇浓度在100μM与50μM、150μM与100μM、200μM与150μM对白念珠菌生物膜抑菌作用的统计学比较存在显著性差异（p<0.05）。不同生物膜时相，法尼醇对白念珠菌耐药株和易感株的抑菌趋势一致。.研究发现法尼醇降低不同生物膜时相易感株和耐药株麦角甾醇水平，与未处理对照组统计学比较有显著性差异（p<0.05）。本研究进而证实不同生物膜时相，白念珠菌生物膜麦角甾醇水平不同；相同的生物膜时相，易感株和耐药株麦角甾醇水平也不同。.激光共聚焦发现法尼醇抑制白念珠菌生物膜形成，法尼醇降低6h、12h、24h和36h白念珠菌易感株生物膜厚度，法尼醇降低6h、12h和24h白念珠菌耐药株生物膜厚度。不同时相生物膜，法尼醇都降低易感株和耐药株生物膜厚度，与未处理对照组统计学比较有显著性差异（p<0.05）。.扫描电镜发现法尼醇对6h和12h白念珠菌生物膜细胞形态存在明显作用。与法尼醇未处理对照组比较，法尼醇处理后易感株和耐药株生物膜菌丝形态减少，酵母细胞增多。