新生隐球菌有性生殖过程中一个新颖的细胞命运决定机制的研究

Sexual reproduction is a universal feature in eukaryotes including fungi. One of the model organisms used to study sexual reproduction is the prevalent human fungal pathogen Cryptococcus neoformans, which is responsible for more than half a million deaths annually. Biologically, sexual reproduction in C. neoformans is critical for coordinating pathogenesis and virulence evolution. C. neoformans possesses two sexual reproduction forms: α-a bisexual reproduction (bisex) and α unisexual reproduction (unisex). Previous studies showed that α mating type cells can undergo both bisex and unisex in response to the same stimulatory cues but can not exert the transition between them as long as either of sexes is initiated. Consistently, parallel studies indicated that these two sexual programs antagonize each other developmentally and genetically. These data support the idea that α mating type cells likely employ an unkown cell-fate decision mechanism directing the specificity of sexual program. This phenomenon is coined by us as sexual program decision (SPD). Here we propose to utilize a systemic approach to explore the transcriptional factors engaged in SPD, elucidate the architecture of SPD regulatory network and identify the master regulator involved. We will also dissect the molecular mechanism of SPD through characterizing the molecular function of key targets of SPD master regulator. The results are expected to 1) ascertain the regulatory basis underlying Cryptococcus sexual reproductions; 2) provide a brand-new insight into how cell fate determination dictates the specificity of sexual reproductions in eukaryotes.

在人类病原真菌中，有性生殖可作为重要生物学策略加速毒力进化进程。其中，新生隐球菌是研究有性生殖的模式病原真菌。该菌每年导致约60万人死亡，致死率超过20%，其有性生殖过程对于感染孢子的产生和毒力进化的推动十分关键，然而其中分子调控机制所知甚少。该菌存在两种交配型α和a ，并主要以α同性生殖和α-a异性生殖两种方式进行有性繁殖。申请人前期研究指出，存在一个未知的细胞命运决定环路帮助新生隐球菌在上述两种性生殖方式之间进行调节和选择，该现象在本项目中被命名为性程序决定。本项目拟采用系统生物学手段完整揭示性程序决定相关调控因子，梳理调控因子构成的网络结构，确定其中起决定作用的核心调控蛋白，并以核心调控蛋白作为切入点解析性程序决定过程关键分子/细胞调控机理。本项研究拟从一个崭新的角度理解新生隐球菌有性生殖发生/调节的分子调控基础，并有助于加深对细胞命运决定在真核生物基础生命繁殖中的关键作用的理解。

有性生殖通过增加子裔的遗传多样性加速病原真菌的毒力进化。新生隐球菌存在两种交配型α和a，可以进行α同性生殖和α-a异性生殖，是研究有性生殖的模式病原真菌，其有性生殖过程对于感染孢子的产生和毒力进化的推动十分关键。.通过CRISPR-Cas9敲除技术将5个性别决定基因全部缺失，本项目研究发现新生隐球菌的同性生殖不依赖于其性别决定，群体密度感应因子Qsp1通过非典型锌指蛋白Cqs2与velvet家族调控蛋白Vea2分别调控了同性生殖与异性生殖过程。在性程序决定方面，通过构建有性生殖过程中呈现动态表达的92个调控因子的缺失突变体文库，对每个缺失突变体对于同性生殖和异性生殖的影响进行评估，构建了新生隐球菌性程序决定的调控网络，确定了由锌指调控蛋白Znf2与细胞周期蛋白Cln1所构成的核心调控环路，解析了其在同性生殖和异性生殖中进行选择的核心机制，为我们对有性生殖性程序决定的认识提供了新的线索，为有性生殖的发生与进化提供新的认识。