Puf4-mediated Metabolic Reprogramming and Virulence Attenuation in Cryptococcus neoformans

Cryptococcus neoformans is a ubiquitous environmental fungal pathogen whose pathogenicity is closely linked to its ability to adapt to host environments. The RNA-binding protein Puf4 plays a key role in regulating the cell wall of C. neoformans. However, the specific mechanisms by which Puf4 regulates metabolism and virulence remain unclear. In this study, we systematically investigated the role of Puf4 in the metabolism and virulence of C. neoformans using RNA sequencing (RNA-seq), biochemical analysis, and in vivo animal experiments. Our results showed that the Puf4-overexpressing strain showed cellular aggregates, suggesting the formation of amyloid-like structures. RNA-seq analysis revealed that Puf4 was enriched in key metabolic pathways, including carbohydrate metabolism, oxidative phosphorylation, sucrose metabolism, and glycolysis. Biochemical analysis revealed that Puf4 overexpression led to a significant increase in total carbohydrate and glycogen content. Additionally, the Puf4-overexpressing strain showed elevated ATP levels, and enhanced resistance to antimycin A, indicating improved oxidative stress tolerance. Notably, Puf4 overexpression resulted in reduced capsule size and decreased L-DOPA production, accompanied by downregulated expression of the capsule-associated gene CAP10 and the melanin biosynthesis gene LAC1. In vivo animal experiments further confirmed that the virulence of the Puf4-overexpressing strain was significantly attenuated. In conclusion, Puf4 plays a key role in regulating C. neoformans adaptation to host environments and pathogenicity. Future research will further validate the functions of Puf4 and explore its potential as a novel antifungal target, providing innovative theoretical insights and targets for the development of antifungal drugs.