白念珠菌转录因子Cas5通过调控Crz1激活钙信号通路的分子机制研究

Candida albicans is able to respond to multiple environmental pressures through a very important calcium signaling pathway. Previous reports have assigned a key role to the transcription factor Crz1 in intracellular calcium homeostasis. However, Crz1 is dispensable for pathogenicity and drug sensitivity of Candida albicans, implying that the calcium signaling pathway requires other unknown factors in regulating fungal virulence and drug sensitivity. By screening our homozygous gene deletion library, we identified a novel factor Cas5 operating in calcium signaling pathway. And this transcription factor is involved in regulation of virulence and drug sensitivity of Candida albicans. In addition, after knockout of Cas5, the expression level of Crz1 protein was significantly decreased, and Crz1 could not enter the nucleus to function under the induction of calcium, which means that Cas5 activates calcium signaling pathway through regulating Crz1. This project is intended to be studied by genetics, functional genomics and molecular biology with two specific aims: 1) Mechanisms of how Cas5 regulates the activation of calcium signaling pathway by interacting with other known factors such as Crz1; 2) Mechanisms of how Cas5-mediated calcium signaling pathway regulates the virulence and drug sensitivity. This study is expected to gain a more comprehensive picture of calcium signaling pathway and we hope that our proposed research can discover new targets of anti-fungal drugs.

钙信号通路是白念珠菌应答环境压力的重要信号途径。虽然Crz1对于真菌胞内钙稳态系统的精细调节至关重要，但基因敲除Crz1并未改变白念珠菌毒力及其药物敏感性，提示钙信号通路参与这两方面的调控尚存其他未知因子。课题组前期工作首次发现转录因子Cas5参与白念珠菌钙信号通路调控，并与该菌毒力和药物敏感性直接相关。此外，敲除Cas5后，Crz1蛋白表达水平显著下降，且在钙离子诱导下Crz1无法进入细胞核发挥功能，表明Cas5可能通过调控Crz1激活钙信号通路。本项目拟通过遗传学、功能基因组学及分子生物学技术等展开研究：1）揭示Cas5如何与Crz1等已知因子相互作用共同调控白念珠菌钙信号通路的激活；2）阐明Cas5介导的钙信号通路如何调控白念珠菌毒力与药物敏感性。该研究对全面解析白念珠菌钙信号调控通路和基于该通路探索新型抗真菌药物靶点具有重要意义。

钙信号通路是白念珠菌应答环境压力的重要信号途径。虽然Crz1对于真菌胞内钙稳态系统的精细调节至关重要，但是基因敲除Crz1并没有改变白念珠菌毒力及其药物敏感性，这提示钙信号通路参与这两方面的调控存在其他未知因子。基因敲除后表型对比显示Cas5和钙调磷酸酶调节亚基Cnb1在白念珠菌应答环境压力、耐受药物和感染小鼠等方面的调控作用类似。转录组测序分析显示白念珠菌中87%的钙离子激活基因是依赖于Cnb1的，76%的钙离子激活基因是依赖于转录因子Crz1的，66%的钙离子激活基因是依赖转录因子Cas5的。依赖于Cas5钙离子激活基因的82.4%是受Crz1调控的，这提示Crz1是白念珠菌细胞内钙离子稳态系统的关键调控因子。滴板实验显示过表达基因CRZ1能够部分回复cas5缺失株对钙离子胁迫和唑类药物的耐受性。同时RT-qPCR和Western blot实验结果显示敲除CAS5后，Crz1的转录水平没有变化，但蛋白表达显著下降。间接免疫荧光实验显示cas5缺失株中Crz1响应细胞外高浓度钙离子时无法进入细胞核。这些结果表明Cas5通过调控Crz1的亚细胞定位和蛋白表达来介导白念珠菌细胞内钙离子稳态。RNA-Seq分析和RT-qPCR实验结果显示钙离子胁迫下，敲除CAS5后Tpk1等多数蛋白激酶转录水平显著升高。而ChIP-Seq实验结果表明钙离子胁迫下转录因子Cas5直接结合到了上述Tpk1等激酶的启动子区域。我们通过Co-IP和间接免疫荧光等实验证实了Nmd5是Crz1的一个核输入蛋白，但我们并没有观察到Cas5对Nmd5转运Crz1进入细胞核的影响。因此，转录因子Cas5是直接抑制Tpk1等激酶的转录来促进Crz1进入细胞核，从而介导白念珠菌细胞内钙离子稳态。综上所述，Cas5是钙调磷酸酶信号通路上介导白念珠菌毒力和药物耐受的重要调控因子。