GST在蜜蜂中肠细胞感染微孢子虫后的调控功能研究

Nosema ceranae is the most common pathogen in adult honey bees. N. ceranae needs to largely modulate host cell functions, and the inhibition of apoptosis is the most obvious phenomenon in the infection. N. ceranae alters various host proteins; among them, glutathione-S-transferase (GST; a group of universal genes in eukaryotes) was increased in the quantities and the activities. However, honey bee GSTs have not been well characterized. GSTs are responsible for various cell function modulations in addition to detoxification in human studies. In cancer studies, cancer cells have upregulated GST activities and lowered apoptosis rate. One human GST, GST P, has been demonstrated to directly inhibit apoptosis. Although GST P does not exist in insects and bees, certain insect GST S and GST D showed highly similar structures to that of GST P. This research proposal plans to identify honey bee GST modulation in apoptosis using N. ceranae infection as the research model. First, we will use proteomic methods to identify the specific GST alterations and modifications resulted from N. ceranae infection. The other approach will be carefully identifying each GST gene expression changes during the infection. The identified GSTs will be interfered using RNAi assay. Additional GST inhibition drugs will be included in the functional assays as well. Finally, the changes will be carefully monitored using GST antibodies and cytochemistry with CLARITY method. Bees with N. ceranae resistance showed much higher rate of apoptosis than regular bees. Our results can annotate honey bee GSTs and identify the GST modulating apoptosis. The same GST could be applied as a drug target to treat N. ceranae infection or enhance the resistance to the disease.

东方蜜蜂微孢子虫为最常见的成年蜜蜂病原，感染会抑制细胞凋亡，增加谷胱苷肽S-转移酶 (GST) 活性。但蜜蜂GST功能缺乏研究报道。研究已表明，人类癌化细胞的GST活性增加，其中GSTP能直接调控细胞凋亡。昆虫虽无GSTP，但有结构相似的GST，而蜜蜂细胞凋亡与GST的关联尚无研究。因此，微孢子虫病抑制中肠细胞凋亡的现象可作为研究蜜蜂GST功能的模型。本研究将明确微孢子虫影响蜜蜂GST的种类、数量，与翻译后修饰；并确认GST互作蛋白。同时，监控感染中蜜蜂GST基因表达；利用RNAi干扰选定的GST基因；再利用药物干扰GST活性，并以免疫组化验证以上试验结果。通过确认微孢子虫、GST与细胞凋亡间的关系，对未来以GST为抗性或药物靶标靶标，让蜜蜂维持细胞正常凋亡，增加抗病性，控制微孢子虫感染。

蜜蜂是最重要的授粉昆虫，然而，蜜蜂却面临许多负面因素与挑战，在多种因素中，微孢子虫病是目前蜜蜂中流行最广泛的一种成蜂疾病，虽不造成蜜蜂急性死亡，但造成许多细致的改变。微孢子虫是一类独特的细胞内寄生真菌，主要感染成年蜜蜂中肠。感染前期没有明显的症状，直到感染后期才有明显的症状或体征。目前对于微孢子虫感染的转录组有多个研究，但蛋白质方面研究较少，所以本项目以蛋白质组学方式，及研究肠道微生物的协力作用等等方向著手，对于目前微孢子虫感染对蜜蜂造成的改变有进一步的解析，其中肠道微生物的研究结果对目前许多尝试使用肠道微生物防治蜜蜂孢子虫病的研究做出了理论性上的解释，也预期了田野实验中看似冲突的结果，同时也证明了应用肠道微生物来防治疾病需要更精确且仔细的进行。另外我们也证明RNAi在应用防治上有更近一步的解析，增加对选定标的基因的认识。结果产出上也发现组织透明化技术能有完整保留肠道微生物在蜜蜂肠道内的分布状况，但分子探针或抗体的标记方式还需要精进，避免伪阳性信号的影响。项目中对蜜蜂中肠蛋白质组的研究，虽然完成，但还在进行投稿准备，部分结果还没有完成分析。