H3K4 methylation regulates development, DNA repair, and virulence in Mucorales. H3K4 methylation regulates development, DNA repair, and virulence in Mucorales

Mucorales are basal fungi that opportunistically cause a fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic organisms. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis. Overall design: RNA-seq experiment to analyze the differences in gene expression caused by Set1 KO in M. lusitanicus. Total RNA was extracted from MU636 and MU1350 (set1 KO) for 24 hours on YPG media (solid) at 26 ºC. For each strain, we performed three biological replicates

毛霉目（Mucorales）是一类基底真菌，可机会性引发致死性感染——毛霉病（mucormycosis，又称黑真菌病）。该疾病因高致死率且近期与SARS-CoV-2感染相关，对人类健康构成严重威胁。另一方面，组蛋白甲基化是一种具有多效性的基因调控机制，可影响多种致病生物的毒力。然而，此前从未有针对毛霉目真菌组蛋白层面表观遗传变化的相关研究。本研究解析了Set1的功能角色：Set1是一种组蛋白甲基转移酶（histone methyltransferase），可催化H3K4位点的甲基化修饰，该修饰与基因转录激活及病原体毒力密切相关。通过对葡萄牙毛霉（Mucor lusitanicus，此前被称为Mucor circinelloides f. lusitanicus）的比较基因组分析，仅发现一个与白色念珠菌（Candida albicans）和酿酒酵母（Saccharomyces cerevisiae）中Set1同源的基因，其包含典型的SET结构域。对set1基因的敲除菌株缺失了H3K4单甲基化、二甲基化与三甲基化的酶促活性。该类菌株的营养生长与产孢能力均显著下降。此外，set1缺失菌株对十二烷基硫酸钠（Sodium Dodecyl Sulfate, SDS）、甲基磺酸乙酯（Ethyl Methanesulfonate, EMS）及紫外线（Ultraviolet Light, UV）更为敏感，表明其细胞壁修复与DNA损伤修复过程存在严重缺陷，提示Set1与这些修复过程密切相关。在病原-宿主互作实验中，set1基因缺失菌株在吞噬体内的极性生长出现缩短，且在体外与体内实验中均表现出毒力减弱。本研究结果表明，组蛋白甲基转移酶Set1可协调葡萄牙毛霉致病相关的多种细胞过程，有望成为未来对抗毛霉病的重要治疗靶点。整体实验设计：通过RNA测序（RNA-seq）实验分析葡萄牙毛霉中Set1敲除（KO）导致的基因表达差异。从野生型菌株MU636与Set1敲除菌株MU1350中提取总RNA，菌株于26℃的YPG固体培养基中培养24小时。每个菌株设置3次生物学重复。