烟曲霉Cys(6)Zn(2)型转录因子LeuB家族调控亮氨酸合成网络和毒力的解析

New broad-spectrum antifungals, which display high effectiveness and reduced toxicity, are required. Since the nutrition of a pathogen during infection is essential for growth and maintenance within the host, new antifungals may be derived from the inhibition of fungal metabolism during pathogenesis. Therefore, targeting of pathways required for nutrient acquisition and metabolism could lead to new antifungals. Since mammals do not have the capacity to produce branched-chain amino acids, the fungal leucine biosynthesis system may be useful as a novel antifungal drug target. .In this project, based on screening T-DNA mutagenesis library, we found that the Cys(6)Zn(2) transcription factor family LeuB (LeuB1, 2 and 3) plays important roles in both leucine biosynthesis and iron regulation. According to BLAST analysis, LeuB1 in A. fumigatus has a deduced 920 amino acids sequence in length and belongs to a Cys(6)Zn(2) transcription factor family, which exclusively exists in fungi with a DNA-binding domain and a specific domain for a transcription factor. Deletion of leuB1 greatly reduces the hyphal growth rates and sharply decreases conidiation in the absence of leucine and/or under the iron starvation condition. These defects could be completely recovered with the addition of both leucine and iron in media. To further address how the leuB family regulates the leucine synthesis pathway and whether leuB family is an important virulence determinant in A. fumigatus, we will construct different strains including single and/or multiple leuB deletions, site-mutated, precise and efficient in-frame integration with GFP and His-tag based on homology-direct integration and a highly efficient CRISPR mutagenesis system. Through phenotypic observation under both liquid and solid cultural conditions, we next further clarify what is the relationship among this putative LeuB family members and how LeuB shows a dual-function transcription factor for both of leucine biosynthesis and the iron acquisition. Thirdly, according to approaches of Chip-seq (Chromatin Immunoprecipitation sequencing) combined with electrophoretic mobility shift assay (EMSA), putative target genes regulated by leuB as a potential transcript factor could be identified. Leucine is not only acts as a substrate for protein metabolism, but can also serve as signaling molecules during signal transduction. In eukaryote, proteasome mediated protein degradation pathway is crucial for the protein metabolism. Thus, we hypothesize that LeuB-deficiency resulted in dramatic protein degradation under iron starvation may be due to impaired leucine biosynthesis which activates the proteasome mediated protein degradation pathway. To verify this hypothesis, SDS-PAGE for whole protein extraction, Western blotting and related biochemistry assays will be carried out under the treatment of the proteasome specific inhibitor MG132 in vivo and in vitro. Finally, virulence tests for different leuB mutants will be carried out in both intranasally and intravenously infected mice model. We propose that the identification of LeuB-targeted genes with a yet uncharacterized link to leucine biosynthesis with iron as well as protein metabolism will aid in finding for new antifungal drug targets required for fungal adaption to host niches and virulence traits.

解析病原真菌特有毒力基因及其调控网络将为新药靶的筛选和利用提供依据。烟曲霉中存在人源缺乏的亮氨酸合成途径，本课题的前期研究从烟曲霉随机突变体库中筛选到该途径中的Cys(6)Zn(2)型转录因子LeuB1，信息学分析表明LeuB家族有三个成员LeuB1,2,3但仅有LeuB1缺失导致低铁敏感、亮氨酸合成受阻、全局性蛋白降解。本课题拟进一步通过烟曲霉CRISPR-Cas9基因编辑技术，构建基因缺失、位点突变和原位加标签的LeuB系列菌株，研究LeuB家族调控亮氨酸合成的分子机制；并结合染色质免疫沉淀(ChIP-seq)、凝胶迁移分析（EMSA）证实LeuB家族转录调控的靶基因、探析LeuB家族及其转录活性与产生动物毒性的关系，进而阐明LeuB家族与亮氨酸合成的网络LeuA和LeuC的调控，解析LeuB家族影响烟曲霉生长发育和毒力的分子功能特征，为新型抗真菌药物设计提供理论依据。

目前的抗真菌药主要靶点是真菌细胞壁和细胞膜，其中的唑类药物是临床一线药物之一。然而，日益加剧的侵袭性真菌感染和不断涌现的耐药菌株导致了可供选择的抗真菌药物的不足，因此，解析病原真菌特有毒力基因及其调控网络将为新药靶的筛选和利用提供依据。本课题的研究发现条件致病菌烟曲霉中存在人源缺乏的亮氨酸合成途径，该途径中的Cys(6)Zn(2)型转录因子LeuB家族有三个候选成员LeuB，B1, B2（现在命名为LeuB, 1, 2），它们均为Zn(II)2Cys6类型的转录因子。本课题按照计划通过烟曲霉CRISPR-Cas9基因编辑技术，构建基因缺失、位点突变和原位加标签的LeuB系列菌株，阐明了LeuB转录因子在亮氨酸合成调控中的功能；并结合染色质免疫沉淀(ChIP-seq)、凝胶迁移分析（EMSA）分析了LeuB的下游靶成员及其功能和调控途径，其中LeuB缺失导致低铁敏感、亮氨酸合成受阻、全局性的蛋白降解和大蜡螟和小鼠动物毒性显著降低（已发表）。另外，在项目执行期间，我们系统筛选了烟曲霉转录因子文库，发现了参与药物易感性以及烟曲霉毒性相关的重要转录因子和相关功能基因 （已发表）。更有意思的是，近期我们意外地发现缺铁环境下添加外源钙导致病原真菌烟曲霉的生长抑制现象，证实了高钙可以抑制真菌对于铁的吸收，从而加剧了铁的缺失，导致真菌生长停止。证实采用铁的螯合剂来降低铁营养的供给，同时结合一定浓度的钙营养，可以显著抑制烟曲霉的生长，这个抑制作用在培养基中和小鼠体内都得到证实。进一步发现高钙和低铁联合调节同样可以抑制病原真菌-白念珠菌和新生隐球菌的生长，这些发现暗示铁限制和高钙营养组合可以是一种新的抗菌策略，这个发现尤其对于目前难以治疗的耐药菌是一种新的策略（专利及待发表数据）。