Hatinguais Remi, Leaves Ian, Brown Gordon D, Brown Alistair J P, Brock Matthias, Peres da Silva Roberta
Medical Research Council Centre for Medical Mycology, University of Exeter , Exeter, United Kingdom.
Fungal Biology Group, School of Life Sciences, University of Nottingham , Nottingham, United Kingdom.
Microbiol Spectr. 2023 Sep 14;11(5):e0507822. doi: 10.1128/spectrum.05078-22.
is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in and . Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene , which is a proposed virulence factor of species. To improve integration of deletion constructs, we deleted the KU homolog that is critical for non-homologous end-joining DNA repair. The use of Δ strains from enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in species. IMPORTANCE Sporotrichosis caused by is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.
是一种新兴的真菌病原体，常与受污染的猫导致的孢子丝菌病的人畜共患病传播有关。在25年内，该疾病不仅在巴西境内传播，如今已蔓延至拉丁美洲的邻国。温度双态性、黑色素、聚糖、黏附素和分泌囊泡与该菌种在哺乳动物宿主中致病的能力有关。尽管已提出某些毒力因子是孢子丝菌病的潜在决定因素，但用于在该菌种中进行反向遗传学研究的分子工具匮乏，严重阻碍了对该疾病潜在机制的剖析。在此，我们证明聚乙二醇介导的原生质体转化是在该菌种和该菌种中进行异源基因表达的有力方法。结合CRISPR/Cas9基因编辑，这种转化方案能够缺失假定的二羟基萘黑色素合酶基因，该基因是该菌种的一种假定毒力因子。为提高缺失构建体的整合效率，我们删除了对非同源末端连接DNA修复至关重要的KU同源物。使用该菌种的Δ菌株可增强转化过程中的同源定向修复，与CRISPR/Cas9结合可增加靶向基因缺失。总之，我们基于CRISPR/Cas9的转化方案为该菌种的靶向基因操作提供了一种有效工具。重要性由该菌种引起的孢子丝菌病是一种需要长期治疗且正在拉丁美洲迅速蔓延的疾病。这种真菌的毒力以及该疾病非典型和更严重表现的激增，使得有必要了解孢子丝菌病的分子机制，以及开发更好的诊断方法和抗真菌疗法。通过开发用于该菌种精确基因操作的分子工具，本研究解决了该菌种稳定基因工程中可靠且可重复工具匮乏的问题，这一直是研究毒力决定因素及其在孢子丝菌病发生过程中作用的主要障碍。
