双组份系统上游调控蛋白及由其介导环境信号的感知在孢子丝菌病发病中的作用

Several signaling cascades including two-component regulatory systems have been discovered to be involved in host environmental response, morphologic switch and pathogensis in dimorphic fungus. In our previous study, DRK1，a two-component protein in sporothrix schenckii was proved to be a soluble histidine kinase lacking transmembrane segments, involving in gene expression regulation in MAPK pathway, spore germination, cell wall composition, melanin formation and inflammatory cells infiltration.However,several questions remain further investigation such as what kind of signals DRK1 senses and whether there exist other upstream transmembrane histidine kinases acting as external signal receptors and interacting with SsDRK1.The following investigations will be carried out in the present research. Part 1: To generate a reporter strain, a sporothrix schenckii strain is transformed via a plasmid contains a transcriptional fusion of the full-length DRK1 promoter in front of the β-galactosidase reporter LacZ. Reporter strain is then transformed with A. tumefaciens strain carrying the binary plasmid.Furthermore,transformants are selected on BHI plates containing X-gal for blue-white color screening at 37 C. Tail-PCR technique is also used to amplify the flank sequences beside T-DNA in white colony strain. Sequences generated from both strands are edited and aligned for a BLAST search in GenBank. At last, data analysis is performed to identify target gene. Part 2: Another reporter strain is made by transforming a sporothrix schenckii strain via a plasmid contains a transcriptional fusion of the full-length target promoter in front of the GFP reporter. And then,fluorescence detection is performed to trace target gene's expression when environmental factor,such as temperature and PH,changes. Finally, lesion infection in murine's model is evaluated when definite environmental signals target gene responds to are changed. This series of study will contribute to elucidate the pathogenesis of sporotrichosis and develop a new method for treatment.

双相真菌通过双组份等信号感受调节系统感知外界环境参数变化而发生形态转化并形成致病性。我们先前研究证实孢子丝菌双组份蛋白DRK1缺乏跨膜结构，是一种位于胞内的可溶性组氨酸激酶，参与对MAPK通路基因表达、孢子萌发、细胞壁组成、黑色素生成及炎细胞浸润的调控。那么，孢子丝菌在入侵宿主后双组份系统感受并传递的是哪种环境信号，又是如何将其传递给胞内DRK1的？本研究将首先构建检测DRK1表达与否的LacZ报告菌株，再以根癌农杆菌 T-DNA 插入突变技术构建报告菌株的突变体库，进一步通过蓝白斑实验筛选出DRK1表达受抑制的突变体并测序，获得DRK1上游调控蛋白Target；构建检测Target表达水平的GFP报告菌株，改变培养基PH值等参数，以荧光显微镜观察Target水平来确定双组份系统感受环境信号的种类，并观察环境信号改变对菌株致病性的影响，为深入揭示孢子丝菌病发病机制及研发新治疗方法奠定基础。

双相真菌通过双组份等信号感受调节系统感知外界环境参数变化而发生形态转化并形成致病性。我们先前研究证实孢子丝菌双组份蛋白DRK1缺乏跨膜结构，是一种位于胞内的可溶性组氨酸激酶，参与对MAPK通路基因表达、孢子萌发、细胞壁组成、黑色素生成及炎细胞浸润的调控。为了进一步明确双组份蛋白感受环境信号的方式和种类，我们首先利用酵母双杂交系统筛选与DRK1相互作用的蛋白，结果鉴定出17个与SsDRK1相互作用的蛋白，其中15个参与DRK1蛋白质合成，转录，转运及降解代谢过程。另外参与细胞内信号传递的蛋白Endonuclease/exonuclease/phosphatase 以及毒力蛋白Prefoldin beta-like protein也被鉴定出来，表明DRK1可能通过这两个蛋白向核内传递信号并参与对孢子丝菌毒力形成进行调控。17个蛋白中并未包含与DRK1相互作用的上游双组分信号蛋白，提示DRK1基因很可能直接感受环境信号的变化，而且此种信号的刺激就来自于胞内。我们进一步通过比较转录组学及生物信息学方法证实孢子丝菌DRK1基因为胞内蛋白，直接感受膜内信号的变化来调控下游Hog1通路基因的表达。尽管DRK1缺乏上游信号传导基因的调控，接下来的研究证实其表达受控于转录调控蛋白AbaA的调节：我们于孢子丝菌蛋白质组库中鉴定了AbaA基因并确定DRK1启动子序列中AbaA蛋白结合位点的存在，进一步构建AbaA干扰菌株发现AbaA基因干扰后孢子丝菌DRK1基因显著下调，证实DRK1基因表达受转录调控蛋白AbaA的正向调控。最后通过观察不同渗透压，氧分压等培养条件的野生株，DRK1干扰株菌落形态，镜下结构，生长曲线及对Fludioxonil 及Iprodione敏感实验等指标证实DRK1感受的环境信号种类为渗透压和氧分压。至此，我们证实SsDRK1基因处于双组分信号通路最上游位置，直接感受胞内渗透压，氧分压的变化，并受转录调控蛋白AbaA的正向调控，为深入揭示孢子丝菌病的发病机制及以DRK1为靶位开发孢子丝菌病新的治疗手段奠定基础。此外，我们对发生于糖尿病患者由Aureobasidium pullulans var. melanogenum导致皮下组织感染的病例进行了首次报告，并复习文献总结了Aureobasidium pullulans所致皮肤及皮下组织感染的病例特点。