Laboratory of Applied Immunology, Campus Darcy Ribeiro, University of Brasíliagrid.7632.0, Asa Norte, Brasília, Federal District, Brazil.
CNRS, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses et Antifongiques, Institut Pasteurgrid.428999.7, Paris, France.
Microbiol Spectr. 2022 Oct 26;10(5):e0150422. doi: 10.1128/spectrum.01504-22. Epub 2022 Aug 25.
Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hyg] or neomycin resistance [Neo]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.
新生隐球菌是一种机会性真菌病原体，以其感染和颠覆吞噬细胞的非凡能力而闻名。这种能力为其在宿主内的生存和持续提供了保障，并依赖于表型可塑性。新生隐球菌的存活但非可培养 (VBNC) 表型最近被描述，对其研究有助于理解隐球菌病的病理生理学。荧光菌株的使用改善了宿主相互作用研究，但仍未得到充分利用。在这里，我们将组蛋白 H3 或多聚（A）结合蛋白 (Pab) 融合到增强型绿色荧光蛋白 (eGFP) 或 mCherry 中，获得了一组具有不同颜色、荧光模式和选择性标记（潮霉素 B 抗性 [Hyg] 或新霉素抗性 [Neo]）的新生隐球菌转化体。我们验证了它们在应激反应、毒力相关表型表达、交配、在大蜡螟中的毒力以及在小鼠巨噬细胞内的存活方面与亲本菌株的相似性。最亮的转化体 PAB-GFP 成功应用于通过流式细胞术和荧光显微镜分析吞噬作用。此外，我们证明了工程化的荧光新生隐球菌菌株能够产生 VBNC 细胞。GFP 标记的 Pab1 是应激反应的关键调节剂，证明了 Pab1 的核保留和细胞质应激颗粒的组装，揭示了与休眠新生隐球菌细胞相关的转录后机制。我们的结果支持 PAB-GFP 菌株是研究新生隐球菌的有用工具。新生隐球菌是一种人类致病性酵母，可进入休眠状态，每年在全球造成超过 18 万人死亡。我们设计了一组荧光转化体来帮助研究新生隐球菌。带有 GFP 标记的 Pab1 的突变体提高了用于宿主相互作用研究的基于荧光的技术。此外，这种突变体诱导了存活但非可培养的表型，并揭示了与休眠新生隐球菌相关的转录后机制。荧光突变体的实验使用可能会揭示新生隐球菌-宿主相互作用和真菌生物学，包括休眠表型。
