白念珠菌Wor1介导的调控网络在白灰形态转换中的作用机制

Candida albicans is the most frequently isolated opportunistic pathogen of humans. C. albicans can switch heritably at high frequency between several phenotypes. The ability of morphologic switching is closely related to its pathogenicity. One of the high-frequency switching systems is white-opaque switching which is related to mating. We isolated and identified Wor1 (white-opaque regulator 1) as a master regulator of white-opaque switching. WOR1 expression displayed an all-or-none pattern. The expression of WOR1 was undetectable in white cells, but it was highly induced in opaque cells, suggesting its role in white-opaque switching. Deletion of the WOR1 blocked opaque cell formation; Over-expression of the WOR1 converted all cells to stable opaque cells. Importantly, the ectopic expressed Wor1 induces the transcription of WOR1 from the WOR1 its own locus, which correlates with the switching to opaque phase. The self-sustaining feedback regulation of WOR1 transcription is necessary and sufficient to promote white-opaque switching. To elucidate the mechanism of Wor1 mediated regulatory network in white-opaque switching, we constructed LexA-Wor1 as bait to screen the genomic library of C. albicans and obtained a series of Wor1 interacting proteins, including Orf19.3345, Orf19.3845, Orf19.6291, Orf19.5035, which are involved in regulation of multiple cellular processes including protein modification, DNA damage response, chromatin remodeling and transcriptional machinery. Our aim in this research will be (1) Identification of the Wor1 mediated regulatory network; (2) Effects of Wor1 modification including sumoylation, phosphorylation and acetylation on white-opaque switching; (3) Roles of the Wor1 mediated regulatory network in transcriptional activation of white-opaque switching genes; (4) Regulation of the interplay between the Wor1 and DNA damage response complex in white-opaque switching; (5) Functional study of Wor1-mediated recruitments of chromatin remodeling complex and chromatin silencing complex in white-opaque switching. Our goal is to understand the molecular and cellular mechanisms of Wor1 mediated regulatory network in white-opaque switching of C. albicans.

白念珠菌Wor1是主控白灰形态转换的一个关键因子，该因子的缺失阻断白菌向灰菌的转换，而该因子的大量表达使细胞锁定在灰菌状态。为了研究Wor1在白灰形态转换中的调控机制，我们利用酵母双杂交系统，鉴定分析了多个Wor1相互作用蛋白，包括Orf19.3345, Orf19.3845, Orf19.6291, Orf19.5035等十多个蛋白因子，序列分析比较表明这些蛋白分别属于蛋白质修饰、DNA损伤修复应答、染色质重塑以及基因转录机器相关的调控因子。本项目研究内容包括：1、Wor1表观遗传调控网络的鉴定和建立；2、Wor1蛋白的修饰包括SUMO化、磷酸化以及乙酰化在激活白菌向灰菌转换过程的功能作用；3、Wor1调控网络在白灰形态转换相关基因转录起始中的激活机制；4、Wor1与DNA损伤应答复合物相互作用在白灰转换中的调控作用；5、Wor1介导的染色质激活和沉默在白菌和灰菌维持中的作用机制。

白念珠菌Wor1是主控白灰形态转换的一个关键因子，该因子的缺失阻断白菌向灰菌的转换，而该因子的大量表达使细胞锁定在灰菌状态。我们利用酵母双杂交系统，鉴定分析了多个Wor1相互作用蛋白，序列分析比较表明这些蛋白分别属于蛋白质修饰、DNA损伤修复应答、染色质重塑以及基因转录机器相关的调控因子。Wos1是一个新的SUMO E3连接酶，通过对Wor1的K385进行SUMO化修饰进而影响其功能，从而响应CO2的刺激调控白灰转换。WOS1基因的缺失降低白菌向灰菌转换的效率，过表达WOS1基因则促进白菌向灰菌的转变。Wos1对白灰转换的促进作用依赖于Wor1，WOS1的缺失延缓了外源表达的WOR1对内源WOR1基因的转录激活以及对白灰转换的促进作用。高浓度CO2能上调WOS1的转录，进而提高Wor1的SUMO化水平，激活白菌向灰菌的转换，CO2调节的WOS1转录依赖Flo8的存在。Siz1也是一个SUMO E3连接酶， SIZ1基因的缺失能够在很多条件下显著地提升白灰转换效率，在高温条件下比野生型菌株形成更多灰菌，而全长的Siz1蛋白是介导高温条件下白菌形成所必需的。在系统感染实验中，siz1的缺失以及过表达均导致白念珠菌毒力下降。Rad9是DNA损伤应答过程中一个重要的DNA检验点蛋白，在DNA损伤试剂MMS存在时，RAD9基因的缺失阻碍了灰菌形成。MMS处理能促进Rad9的磷酸化，并激活下游蛋白激酶Rad53的超磷酸化，Rad9同时介导Wor1与Rad53的相互作用，从而把Wor1的调控与Rad53信号途径相联系。肠道感染实验表明，rad9缺失株的毒力显著下降。Fun30是一个新的染色质重塑因子，具有Snf2家族保守的结构域ATPase和HSA，过表达Fun30促进CO2条件下白菌向灰菌的转换，同时也能促进空气条件下灰菌向白菌的转换， 而FUN30基因的敲除延迟CO2条件下白菌向灰菌的转换，同时也延迟空气条件下灰菌向白菌的转换。Fun30能够被Wor1蛋白招募到染色质位点，Fun30与WOR1启动子区的结合依赖Wor1的存在，同时Fun30的缺失会降低Wor1与染色质结合位点的能力。Hpc2是一个组蛋白伴侣分子，过表达Hpc2抑制白菌向灰菌的转换，Hpc2的缺失则促进灰菌的形成。Hir1能招募Hpc2并组装成HIR复合物，结合于染色质进一步调控核小体的结构。