摘要:在自然環境中,光為一主要的能量來源,亦是調控生物(包含真菌)的生理、發育及行為的重要環境因子。隱球菌(Cryptococcus neoformans),為一異宗交配型之擔子菌,伺機性人體病原真菌,其具有演化保守的光反應調控基因CWC1及CWC2,能感受藍光,抑制有性生殖雙核菌絲之形成與生長。為進一步探討藍光抑制雙核菌絲之分子機制,尋找隱球菌Cwc蛋白質之下游基因,本實驗室利用農桿菌突變技術進行suppressor 篩選,尋找具有回復CWC1大量表現株抑制生殖菌絲生長之突變株。目前已篩選到回復不同程度菌絲生長之農桿菌T-DNA轉殖株,其中4株回復最多菌絲生長之突變株,T-DNA插入破壞之基因為CWC2,此發現印證suppressor 篩選之有效性。其中,並選擇回復較多菌絲生長或伴隨有其他性狀之突變株,進一步界定T-DNA插入所破壞之基因,發現這些基因包括,mediator基因SSN8,酵母菌出芽生殖位置調控基因BUD16及 MUB1,減數分裂調控基因IME2/CRK1等。SSN8基因,又名SRB11/UME3/CYCC/RYE2,為mediator複合蛋白質之一員,啤酒酵母菌的研究顯示,其廣泛參與包括糖的利用、減數分裂,細胞週期等生理調控。出芽生殖調控基因BUD16,蛋白質產物為吡多醛激酶 (pyridoxal kinase),於維生素B6生合成路徑中,將吡多醛 (pyridoxal) 轉變成生物可利用的5’-磷酸吡多醛 (pyridoxal 5’-phosphate, PLP)。IME2/CRK基因,經由IME2基因的活化,共同參與減數分裂的前期調控。為瞭解這些基因在隱球菌中,藍光抑制有性生殖菌絲形成,及其他生理的角色,已於野生型及CWC1大量表現株基因突變株之構築,有性生殖性狀的研究顯示,這些基因在隱球菌有性生殖菌絲之形成上,皆扮演負調控之角色。而ssn8突變株之研究更發現,SSN8基因亦參與侵入菌絲生長,致病因子黑色素及莢膜之形成,以及細胞壁完整性等生理調控。本3年期研究計劃之主要目標包括,1)找尋直接受Cwc1/Cwc2轉錄調控之隱球菌下游基因,2)探討隱球菌mediator蛋白質Ssn8如何參與有性生殖與單核菌絲生長、致病性,以及其他生理之調控;Ssn3 (cyclin-dependent protein kinase)是否亦共同參與相關生理之調控,3) 界定酵母菌出芽生殖位置調控蛋白質Bud16及 Mub1於隱球菌光反應、出芽生殖位置調控及菌絲生長之角色,4)瞭解減數分裂Crk1蛋白質,如何參與隱球菌有性生殖與減數分裂之調控。希望經由本計畫之研究,能進一步建構出CWC1/CWC2如何協同其他基因,進行隱球菌光反應、菌絲生長及Ssn8於致病性生理之調控。
Abstract: Light is the major source of energy on Earth and also an important environmental signal which regulates the physiology, development and behavior of many organisms including fungi. Cryptococcus neoformans, a heterothallic basidiomycetous yeast, can sense blue light and negatively regulate the production of sexual filaments via the evolutionally conserved Cwc1 and Cwc2 proteins. To dissect this photo-inhibition pathway, we conducted a suppressor screen utilized Agrobacterium tumefaciens-mediated transformation (ATMT) technique to identify mutants suppressing the mating phenotype of the CWC1 overexpression strain, which displayed no filaments under light illumination. Mutants which suppressed the light-dependent CWC1 overexpression phenotype and restored different levels of mating filamentation were identified. Four suppressor strains which restored the most abundant filaments were found that T-DNAs were inserted at the CWC2 locus, which validated our suppressor screening. In addition, mutants restoring high level of filaments or showing other phenotypes have been selected for further characterization. Cryptococcus neoformans genes disrupted by T-DNA insertion among these mutants include the mediator gene SSN8, budding regulatory genes BUD16 and MUB1, meiosis regulator gene IME2/CRK1. SSN8, also named SRB11/UME3/CYCC/RYE2, is a member of Mediator complex which is widely involved in the physiological processes, including sugar utilization, meiosis, and cell phase control. The Bud16 protein is a predicted pyridoxal kinase which converts pyridoxal into pyridoxal 5’-phosphate (PLP), the biologically active form of vitamin B6. The Ime2/Crk1 protein, activated by Ime1, is an early meiosis-specific gene. Gene specific deletion mutation of some of these genes in the wild-type and CWC1 overexpression strains have been made. Phenotypic characterization consistently suggests that these genes negatively regulate mating process. Interestingly, SSN8 has been found to involve in the suppression of invasive growth, melanin and capsule formation and required for maintenance of cell integrity. The goals of this three-year grant proposal are divided into the following aims. The first aim is to identify the genes directly controlled by the Cwc1/Cwc2 complex in the light-regulated sexual differentiation in C. neoformans. The second aim is to determine how the Ssn8 mediator regulates mating, haploid filamentation, virulence and other physiological pathways in C. neoformans and if the cyclin-dependent protein kinase Ssn3 also plays similar roles in these process. The third aim is to characterize how budding genes Bud16 and Mub1 regulate the light-repressed filamentation pathway in C. neoformans. The final aim is to determine how the early meiosis gene Crk1 regulates sexual development in C. neoformans. To the end, we hope to generate a network scheme describing how CWC1/CWC2 and these genes transduce the light signal to negatively regulate mating, monokaryotic filamentation and how Ssn8 regulates virulence other processes in C. neoformans.