Castelli Rafael F, de Oliveira Haroldo C, Santos Marlon D M, Camillo-Andrade Amanda C, Dos Reis Flavia C G, Carvalho Paulo C, Rodrigues Marcio L
Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil.
Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil; Analytical Biochemistry and Proteomics Unit, IIBCE/Institut Pasteur de Montevideo, Uruguay.
J Proteomics. 2025 Aug 15;319:105483. doi: 10.1016/j.jprot.2025.105483. Epub 2025 Jun 19.
In the fungus Cryptococcus neoformans, the aminophospholipid translocase 1 (Apt1) flippase plays roles in virulence, membrane architecture, and extracellular vesicle (EV) polysaccharide cargo. The effect of APT1 deletion on the fungal proteome is unknown, limiting the understanding of its functions in physiology. The APT gene family also includes APT2, whose functions in C. neoformans are virtually unknown. We investigated the impact of APT1 and APT2 deletion on EV formation in C. neoformans. The absence of Apt1, but not Apt2, led to a decreased concentration of the polysaccharide glucuronoxylomannan in EVs. We characterized the EV and cellular proteomes of C. neoformans mutants lacking APT1 and APT2, comparing them to the proteomes of wild-type (WT) cells. Paired comparisons revealed that WT and mutant EVs shared a significant part of their cargo but showed several strain-specific molecules and exhibited major differences in the abundance of various proteins. Conversely, the cellular proteomes of both mutants largely overlapped with WT (95.4 % shared proteins. Protein network analyses highlighted mutant-specific shifts: the apt1Δ/apt2Δ proteomes converged on secondary metabolite biosynthesis and RNA metabolism clusters, diverging from the predominance of translation in WT cells. These findings establish APT1 and APT2 as key regulators of EV composition in C. neoformans. SIGNIFICANCE: Our study reveals that the aminophospholipid translocase 1 (Apt1) and aminophospholipid translocase 2 (Apt2) play distinct roles in the physiology of Cryptococcus neoformans, particularly in the formation and composition of extracellular vesicles (EVs). By demonstrating that Apt1 deletion alters the proteomic landscape and reduces glucuronoxylomannan levels in EVs, while Apt2 deletion has no such effect, our findings provide critical insights into the functional divergence of these flippases. These insights underscore the potential of Apt1, but not necessarily Apt2, as therapeutic targets for developing novel antifungal strategies against this significant human pathogen.
在新型隐球菌中，氨基磷脂转位酶1（Apt1）翻转酶在毒力、膜结构和细胞外囊泡（EV）多糖货物中发挥作用。APT1缺失对真菌蛋白质组的影响尚不清楚，这限制了对其在生理学中功能的理解。APT基因家族还包括APT2，其在新型隐球菌中的功能几乎未知。我们研究了APT1和APT2缺失对新型隐球菌中EV形成的影响。Apt1的缺失而非Apt2的缺失导致EV中多糖葡糖醛酸木甘露聚糖的浓度降低。我们对缺乏APT1和APT2的新型隐球菌突变体的EV和细胞蛋白质组进行了表征，并将它们与野生型（WT）细胞的蛋白质组进行了比较。配对比较显示，WT和突变体EV共享了很大一部分货物，但显示出几种菌株特异性分子，并且在各种蛋白质的丰度上表现出主要差异。相反，两个突变体的细胞蛋白质组在很大程度上与WT重叠（95.4%的共享蛋白质）。蛋白质网络分析突出了突变体特异性变化：apt1Δ/apt2Δ蛋白质组集中在次生代谢物生物合成和RNA代谢簇上，与WT细胞中翻译占主导地位不同。这些发现确立了APT1和APT2作为新型隐球菌中EV组成的关键调节因子。意义：我们的研究表明，氨基磷脂转位酶1（Apt1）和氨基磷脂转位酶2（Apt2）在新型隐球菌的生理学中发挥着不同的作用，特别是在细胞外囊泡（EV）的形成和组成方面。通过证明Apt1缺失会改变蛋白质组格局并降低EV中的葡糖醛酸木甘露聚糖水平，而Apt2缺失则没有这种效果，我们的发现为这些翻转酶的功能差异提供了关键见解。这些见解强调了Apt1作为开发针对这种重要人类病原体的新型抗真菌策略的治疗靶点的潜力，但不一定是Apt2。
