The role of uncharacterized protein Taf1 in regulating ATP levels and virulence of Cryptococcus neoformans

Abstract

The human fungal pathogen Cryptococcus neoformans poses significant health risks, particularly to immunocompromised individuals, such as those with HIV/AIDS. In this study, we investigate the role of an uncharacterized protein, Taf1, in regulating ATP levels and virulence in C. neoformans. Our previous proteomic analyses confirmed the expression of Taf1, encoded by the gene CNAG_04232. We found that the deletion of the TAF1 gene resulted in the upregulation of 204 genes and the downregulation of 908 genes. Gene Ontology analysis indicated that these regulated genes are associated with metabolic and cellular processes, as well as ATP-dependent activities. Notably, the TAF1-deficient mutant exhibited impaired growth at elevated temperatures (39°C). Furthermore, in a murine model of infection, mice inoculated with the taf1Δ mutant demonstrated significantly improved survival compared to those infected with the wild-type strain, suggesting a critical role for Taf1 in virulence. Additionally, KEGG pathway analysis of RNA-Seq and metabolomics data revealed significant alterations in fatty acid biosynthesis and degradation pathways following TAF1 deletion. Collectively, these findings underscore the essential role of Taf1 in modulating cellular energy and its implications for the virulence of C. neoformans, thereby paving the way for potential therapeutic strategies targeting this pathogen.

Keywords: Cryptococcus neoformans; ATP; Taf1; Virulence.

Fig. 1

Taf1 is essential for thermotolerance and virulence. A To assess the thermotolerance, spot assays were performed on YPD medium and L-DOPA plates, examining the response of each strain at 30°C, 37°C, or 39°C. Specifically, L-DOPA plates were utilized to evaluate melanin production in the various strains. B A total of ten female BALB/c mice were subjected to intranasal inoculation with 10⁵ cells of either the WT strain (KN99) or the taf1Δ mutant strain. To assess survival outcomes, the log-rank Mantel-Cox test was employed for statistical evaluation between the two groups of mice. The analysis revealed significant differences in survival rates between those infected with the WT strain and those infected with the taf1Δ mutant strain (*, P < 0.05)

Fig. 2

The impact of Taf1 loss on gene and metabolite expression. A Bar charts show the number of differentially expressed genes: up-regulated (red) and down-regulated (blue) with a false discovery rate (FDR) of less than 0.05 and a log 2 fold change greater than 1 or less than − 1. B Bar charts display the number of differentially expressed metabolites: up-regulated (red) and down-regulated (blue), both with FDR < 0.05. The efficiency of metabolite identification is shown for negative ion mode (NEG) and positive ion mode (POS)

Fig. 3

The Gene Ontology (GO) categories for differentially expressed genes were identified through RNA-seq analysis of the taf1Δ mutant, utilizing three biological replicates

Fig. 4

Significant changes in intracellular ATP levels and mitochondrial membrane potential were observed following the knockout of the TAF1 gene. Statistical analysis was performed using a Student’s t-test with three biological replicates. Data are presented as mean ± standard deviation. Asterisks indicate statistical significance: *, P < 0.05; ***, P < 0.001. Figures were created using Origin software

Fig. 5

The KEGG enrichment analysis reveals the top 30 pathways enriched for differentially expressed genes, based on three biological replicates

Fig. 6

The KEGG enrichment analysis of metabolites with differential expression, highlighting the top 30 enriched pathways identified through through negative ion mass spectrometry (NEG) and positive ion mass spectrometry (POS), based on three biological replicates