光滑念珠菌棘白菌素类药物耐药株的出现给临床治疗带来严峻挑战，但其耐药发生发展的机制尚不清楚。前期研究发现，光滑念珠菌在形成稳定耐药前存在棘白菌素耐受状态；利用CRISPR/Cas9技术敲除了标准株的RNA结合蛋白Ssd1，发现敲除株对棘白菌素的耐受性降低，且细胞壁几丁质含量减少。我们推测：Ssd1通过调控细胞壁多糖合成影响光滑念珠菌棘白菌素耐受，促进形成稳定耐药。本项目将1）对Ssd1敲除株、回复突变株和过表达株进行体外诱导试验，分析Ssd1对光滑念珠菌棘白菌素耐药突变率的影响；2）比较各菌株在棘白菌素刺激下，细胞壁不同多糖成分含量和多糖合成酶活性及表达量的差异；3）利用RNA结合蛋白免疫共沉淀串联高通量测序技术，筛选并验证与Ssd1结合的RNA，探索Ssd1调控网络，以期明确Ssd1在光滑念珠菌棘白菌素耐受中的分子机制。本研究将为控制耐受发生发展、遏制真菌耐药和研发新药提供新的理论依据。

The emergence of echinocandin resistant in Candida glabrata has posed a serious challenge to clinical treatment. However, the mechanisms of its occurrence and development is still unknown. In the previous study, we found that C. glabrata had an echinocandin tolerance state before resistance. The RNA binding protein Ssd1 of type strain was successfully knocked out by CRISPR/Cas9. The echinocandin tolerance of ssd1Δ was decreased, and the chitin content of cell wall was reduced. We proposed that Ssd1 plays a significant role in the formation of echinocandin tolerance by regulating cell wall polysaccharide synthesis in C. glabrata, and promotes the development of drug resistance. Firstly, we will conduct in vitro induction experiment on ssd1Δ, rescued and overexpressed mutants to analyze the effect of Ssd1 on echinocandin resistant mutation rate in C. glabrata. Then we will compare the content of different polysaccharide components in the cell wall, and the activity and expression level of polysaccharide synthase in each strain under the stress of echinocandin. Finally, our research will screen and verify the RNAs bound to Ssd1 by RNA binding protein immunoprecipitation with RNA sequencing technology to explore the Ssd1 regulatory network and reveal the molecular mechanisms of Ssd1 in C. glabrata echinocandin tolerance. This study will provide new theoretical information on controlling tolerance formation, reducing clinical resistance and developing new antifungal drugs.