新生隐球菌是一种机会性酵母样病原体,主要感染免疫功能低下的个体并引起致命的脑膜炎。有性生殖可以促进不同菌株之间的遗传物质交换。这是导致新型梭菌高致病性耐药菌株出现的原因之一。尽管关于隐球菌有性生殖的调控机制已经做了很多研究,泛素-蛋白酶体系统对隐球菌有性生殖调控的研究较少。这项研究鉴定了一种F-box蛋白,Cdc4,其中包含一个推定的F盒结构域和八个WD40结构域。表达模式分析显示,CDC4基因在新生梭菌的各个发育阶段均有表达,Cdc4蛋白定位于隐球菌细胞核中。体外应激反应实验表明,CDC4过表达菌株对SDS和MMS敏感,但对刚果红不敏感。暗示Cdc4可能调节细胞膜完整性和DNA损伤的修复。真菌毒力实验表明,虽然cdc4Δ突变体生长正常,能产生典型的囊、黑色素等毒力因子,cdc4Δ突变体在小鼠全身感染模型中完全失去致病性。真菌交配实验表明,Cdc4对于新生梭菌的真菌有性生殖也是必不可少的。尽管在交配过程中观察到正常的交配菌丝,在cdc4Δ突变体之间的双侧交配中,担子孢子的产生被阻断。真菌核发育分析表明,在cdc4Δ突变体的双侧交配过程中,在基底内融合后,核未能进行减数分裂,表明Cdc4对隐球菌交配过程中调节减数分裂至关重要。总之,我们的研究表明,F-box蛋白Cdc4对于新生梭菌的真菌毒力和有性生殖至关重要。
Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the
CDC4 gene was expressed in various developmental stages of C. neoformans, and the
Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the
CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that
Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that
Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores\' production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.