格特隐球菌(Cryptococcus gattii)是一种担子菌酵母，是人类隐球菌病的主要致病菌之一，可以感染免疫功能正常的宿主并导致肺部感染、脑膜炎等疾病。近年来，在我国中东部地区，格特隐球菌引发隐球菌病的病例多有报道，主要以VGⅡ和VGⅠ分子型菌株为主。格特隐球菌经肺部侵入，而后可以感染人体几乎所有器官，有中枢神经系统倾向性。其侵染能力主要得益于人体生理温度下的生存能力、漆酶的表达和黑色素的产生、荚膜的形成等毒力因子。然而，随着抗真菌药物的广泛使用，临床上逐渐出现了耐药性菌株。在解析耐药机制、筛选新的药物靶点过程中，转录因子的功能令人关注。

课题组前期发现并鉴定了Atm1在格特隐球菌毒力和致病性中的重要作用。Atm1是位于线粒体膜的转运蛋白，介导铁硫簇自线粒体基质流向细胞质基质，其缺失会引起菌株荚膜厚度显著降低、氧化压力应激能力显著减弱等表型，对格特隐球菌毒力因子产生和细胞生命活动有广泛影响，但其中间途径和作用机制并不清晰。转录组测序结果表明，格特隐球菌G6菌株中Atm1缺失导致CNBG_4950和CNBG_9543基因表达量显著降低，推测CNBG_4950和CNBG_9543的表达受到Atm1激活。生物信息学分析显示CNBG_4950和CNBG_9543分别编码FZC家族和BZP家族的转录因子，根据隐球菌的命名原则分别命名为CAF1和GCN4。Caf1和Gcn4两个转录因子是否在Atm1下游通路中起重要作用从而介导Atm1对菌株表型和毒力的影响是本研究拟解决的关键科学问题。

首先，我们使用荧光实时定量PCR技术对C. gattii G6 atm1Δ突变株中CAF1和GCN4的转录水平进行了验证，结果与转录组测序数据保持一致，说明CAF1和GCN4表达直接或间接的受到Atm1的诱导。Caf1属于FZC家族，是真菌特有的转录因子。我们使用课题组构建的格特隐球菌All-In-One CRISPR-Cas9基因敲除系统在临床菌株G6和标准菌株R265中分别敲除了CAF1。荚膜厚度的测量结果显示，Caf1的缺失会导致C. gattii G6的荚膜厚度明显减小，且在C. gattii R265中观察到了同样的结果，这说明Caf1参与介导Atm1对荚膜形成的影响，且此通路存在一定保守性，随后CAF1的回补实验再次验证了这一点。同时，当Caf1缺失时，C. gattii G6漆酶的形成显著增多且黑色素生成的脱抑制受到阻碍。滴板实验结果表明，C. gattii G6 caf1Δ细胞壁完整性受到破坏，且突变株对渗透压力的应答能力减弱，但对表面活性剂压力抗性和RAPA抗性有所增强。

GCN4编码BZP家族转录因子，系统发育树的结果显示Gcn4在进化过程中保守性较低。同样地，我们同时在G6和R265菌株中构建了GCN4缺失突变株。随后的表型实验结果显示，Gcn4缺失导致C. gattii G6荚膜厚度显著降低，说明Gcn4参与调控C. gattii G6荚膜形成过程。同时C. gattii G6 gcn4Δ对渗透压力的应答能力有所增强。当缺失铁离子时，其菌落大小会显著变小，当补充铁离子时，菌落大小有所恢复。但这些现象在C. gattii R265 gcn4Δ上并没有观察到。因此，我们推测GCN4在C. gattii G6中存在独特的进化途径，Gcn4的功能与离子应激相关，相关细节有待进一步研究。

综上，本研究将转录组测序与CRISPR-Cas9基因编辑技术相结合，对Atm1调控格特隐球菌表型和毒力的分子机制进行研究。研究结果表明，Caf1和Gcn4作为Atm1的下游转录因子，其表达受到Atm1的调控，其中Caf1参与调控荚膜形成，且该功能在临床菌株G6和标准菌株R265中具有保守性。Gcn4主要参与C.gattii G6中荚膜形成和离子相关生物学过程，在标准菌株R265中的作用较小。

Cryptococcus gattii is a type of basidiomycete yeast that is one of the main pathogenic bacteria of human cryptococcosis. It can infect hosts with normal immune function and lead to diseases such as lung infections and meningitis. In recent years, in the central and eastern regions of China, there have been many reports of cases of cryptococcosis caused by C.gattii, mainly with VGII and VGI lineage strains. C.gattii invades through the lungs and can invade almost all organs of the human body, with a tendency towards the central nervous system. Its infection ability mainly benefits from the survival ability under human physiological temperature, the expression of laccase, the production of melanin, the formation of capsule and other virulence factors. However, with the widespread use of clinical drugs, drug-resistant strains have gradually emerged. In the process of analyzing the mechanism of drug resistance and screening new drug targets, the function of transcription factor is of great concern.

We previously discovered and identified the important role of Atm1 in the virulence and pathogenicity of C.gattii. Atm1 is a transport protein located in the mitochondrial membrane, which mediates the flow of iron sulfur clusters from the mitochondrial matrix to the cytoplasmic matrix. Its absence will lead to a significant reduction in the thickness of the capsule of the strain, a significant reduction in the ability to oxidative stress and other characteristics. It has a wide impact on the virulence factor and biological phenotype of C.gattii, but its intermediate pathway are not clear. Therefore, in the analysis of results of RNA-seq to C.gattii G6 atm1Δ, we screened and identified two genes stimulated by Atm1: CNBG_4950 and CNBG_9543. Bioinformatics analysis shows CNBG_4950 and CNBG_9543 encodes transcription factors of the FZC family and BZP family, respectively. They are named CAF1 and GCN4 according to the naming principles of Cryptococcus. Whether these two genes play an important role in the downstream pathway of Atm1 and mediate the impact of Atm1 on the phenotype of the strain is the question that we want to study.

Firstly, we used qPCR to detect the transcription level of CAF1 and GCN4 in C.gattii G6 atm1Δ. The results were consistent with the results of RNA-seq, indicating that the expression of CAF1 and GCN4 was directly or indirectly induced by Atm1. CAF1 encodes a fungal specific transcription factor belong to the FZC family. We used the All-In-One CRISPR-Cas9 gene knock-out system constructed by our research group for C.gattii to knock out CAF1 in C.gattii G6 and C.gattii R265. The measurement results of capsule thickness show that the absence of Caf1 can lead to significant decrease of the capsule thickness of C.gattii G6. The same results were observed in the C.gattii R265, indicating that Caf1 is involved in mediating the effect of Atm1 on capsule formation, and this pathway is somewhat conserved. Subsequently, the compensatory experiment of CAF1 confirmed this conclusion again. Meanwhile, when Caf1 is missing, the formation of laccase increased significantly and the disinhibition of melanin production was blocked in C.gattii G6. In C.gattii G6 caf1Δ, the resistance to cell wall pressure and osmotic decreased, but the surfactant pressure resistance and RAPA resistance increased.

Gcn4 is a transcription factor belonging to the BZP family that only has homologous proteins in Cryptococci. The phylogenetic tree results show that Gcn4 is less conserved during evolution. Similarly, we constructed C.gattii G6 gcn4Δ And C.gattii R265 gcn4Δ.The subsequent phenotype experiments of the strain showed that the absence of Gcn4 would lead to the significant decrease in capsule thickness of C.gattii G6, indicating that Gcn4 participates in the process of capsule formation in C.gattii G6. Simultaneously, the ability of C.gattii G6 gcn4Δ of osmotic pressure stress was enhanced. When iron ions are missing, the colony size will significantly decrease, and when iron ions are supplemented, the colony size will recover. But these phenomena don’t occur in C.gattii R265 gcn4Δ. Therefore, we speculate that a unique evolution of GCN4 in C.gattii G6, and the function of Gcn4 is related to ion stress. Further research is needed on the relevant details.

 In summary, this study combined RNA-seq with CRISPR-Cas9 genome editing technology to study the molecular mechanism of Atm1 regulating the phenotype and toxicity of C.gattii. The research results indicate that Caf1 and Gcn4 are downstream transcription factors of Atm1, and their expression is regulated by Atm1. Among them, Caf1 is involved in regulating capsule formation, and this function is conserved in clinical strain C.gattii G6 and standard strain C.gattii R265. Gcn4 mainly participates in capsule formation and ion related biological processes in C.gattii G6, with a smaller role in C.gattii R265.