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.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12866-025-04207-w.

   Keywords: Cryptococcus neoformans, Taf1, Virulence, ATP

Introduction

   The opportunistic fungal pathogen Cryptococcus neoformans poses a
   significant threat to individuals with compromised immune systems,
   particularly those living with HIV/AIDS [[36]1, [37]2]. Annually,
   approximately 180,000 cases of cryptococcal meningoencephalitis are
   reported, with this fungus responsible for 15% of all AIDS-related
   fatalities [[38]3, [39]4]. The World Health Organization’s recent
   recognition of C. neoformans as a critically important fungal pathogen
   underscores the urgent need for further investigation into the serious
   diseases it causes, characterized by a lack of effective treatment
   options and the absence of a vaccine [[40]5, [41]6]. This substantial
   disease burden highlights the pressing necessity to elucidate the
   mechanisms that facilitate fungal proliferation within vertebrate
   hosts, which is crucial for the discovery of new therapeutic agents and
   vaccine targets [[42]7].

   A key factor influencing the virulence of C. neoformans is adenosine
   triphosphate (ATP), which serves as the primary energy currency for
   cells [[43]8, [44]9]. ATP is crucial not only for cellular metabolism
   but also for various processes that enable C. neoformans to thrive and
   cause disease within the host [[45]10, [46]11]. Research has shown that
   disruption of ATP biosynthesis results in a complete loss of virulence
   in C. neoformans. For instance, Blundell et al. demonstrated that the
   loss of adenylosuccinate synthetase (AdSS) leads to adenine auxotrophy
   and a consequent reduction in virulence in murine models [[47]9]. This
   study highlighted distinct structural differences between fungal AdSS
   and its human counterpart, positioning AdSS as a promising target for
   antifungal drug development [[48]9]. The relationship between ATP
   levels and virulence in C. neoformans is particularly significant, as
   the C. neoformans ability to adapt to diverse environmental stresses
   encountered within the host is central to its pathogenicity. Elevated
   ATP levels are associated with enhanced growth and survival under
   hostile conditions, enabling the pathogen to effectively counteract
   immune challenges [[49]8, [50]12]. Conversely, disruptions in ATP
   production can lead to metabolic dysregulation, thereby impairing the
   C. neoformans ability to sustain its virulence [[51]9, [52]11]. The
   regulation of ATP levels is intricately linked to various metabolic
   pathways, particularly those involved in the synthesis and degradation
   of fatty acids [[53]13]. Fatty acids are essential components of
   cellular membranes, playing a critical role in maintaining membrane
   integrity, facilitating signaling processes, and serving as energy
   reserves. Moreover, they significantly impact the modulation of
   virulence traits in fungal pathogens, including C. neoformans, by
   influencing energy production and cellular responses to environmental
   stressors [[54]14–[55]16].

   In this study, we characterized the role of the uncharacterized protein
   Taf1 in regulating ATP levels and its implications for the virulence of
   C. neoformans. Our findings indicate that the TAF1-deficient mutant
   (taf1Δ) exhibited growth deficiencies at 39°C, probably resulting in
   diminished virulence in a murine model of cryptococcosis. Importantly,
   the TAF1-deficient mutant demonstrated reduced ATP levels compared to
   wild-type (WT) cells. Insights from transcriptional profiling using RNA
   sequencing (RNA-Seq) and metabolomics further support the hypothesis
   that Taf1 regulates ATP levels, likely through pathways involved in
   fatty acid biosynthesis and degradation. Overall, these data
   highlighted Taf1 as a probable contributor to ATP levels and the
   virulence of C. neoformans, establishing its significance in the
   context of fungal pathogenesis and energy metabolism.

Materials and methods

Strain and media

   This investigation utilized two strains: the Cryptococcus neoformans
   var. grubii KN99α WT strain and the mutant strain taf1Δ which carries
   the gene deletion of CNAG_04232 sourced from the Fungal Genetics Stock
   Center (FGSC). To explore the differences in mRNA and metabolomic
   profiles between the WT and the mutant strain taf1Δ, cells were
   cultured in YPD medium (2% dextrose, 1% yeast extract, 2% peptone) for
   around 16 h at 30°C. Following this incubation period, the cultures
   were harvested and washed twice with distilled water. The resulting
   cell pellets were subsequently frozen in liquid nitrogen and stored at
   −80°C for RNA-seq and metabolomics studies. Three biological replicates
   were performed.

Melanin production

   To investigate melanin production, L-DOPA agar plates containing 0.7 mM
   L-3,4-dihydroxyphenylalanine (L-DOPA), 0.1% L-asparagine, 0.1% glucose,
   0.025% MgSO4 · 7H[2]O, 0.3% KH[2]PO[4] and 2% agar (pH 5.6) were
   utilized. C. neoformans cells were spotted on L-DOPA agar plates and
   incubated for 2 days at temperatures of 30°C, 37°C, or 39°C before
   capturing images of the results.

Serial spot dilution assays

   Fungal cultures grown overnight were washed twice with PBS, followed by
   adjusting the cell concentration to 2 × 10^5 cells per ml. After this
   adjustment, 10-fold serial dilutions were created, and 5 µl from each
   dilution—spanning from 10^5 to 10^0 cells—was spotted on YPD agar
   plates (2% dextrose, 1% yeast extract, 2% peptone, 2% agar) or L-DOPA
   agar plates. The agar plates were subsequently incubated at
   temperatures of 30°C, 37°C, or 39°C for a duration of 2 days before
   capturing images of the results.

RNA-seq

   To extract RNA utilizing the Omega Plant RNA Kit (Omega Bio-Tek,
   Catalog No. R6827), approximately 100 mg of the fungal cell sample was
   ground in a mortar with liquid nitrogen, and the resulting powder was
   transferred into a 1.5 mL tube. Then, 500 µl of Buffer RCL, including
   β-mercaptoethanol, was added, and the mixture was vortexed thoroughly.
   The sample was incubated in a water bath at 55°C for 3 min, followed by
   centrifugation at 14,000 g for 5 min to separate the phases. The
   supernatant was carefully collected and transferred to a gDNA Filter
   Column in a collection tube, and centrifugation was performed again at
   14,000 g for 2 min. Next, the supernatant was mixed with an equal
   volume of Buffer RCB by pipetting up and down 5 to 10 times. This
   mixture was divided in half; one portion was transferred to the Hibind
   RNAMini column and centrifuged at 10,000 g for 1 min, discarding the
   flow-through, then the column was returned to the collection tube. This
   process was repeated with the second half of the solution.
   Subsequently, 400 µl of RWC was added to the column and centrifuged at
   10,000 g for 1 min, discarding the flow-through. Then, 500 µl of RNA
   Wash Buffer II was added, and centrifugation was repeated at 10,000 g
   for another minute, with the waste discarded once more. After washing,
   the column was centrifuged at 10,000 g for 2 min to remove residual
   buffers. The Hibind RNAMini column was transferred to a new 1.5 mL
   RNase-Free tube, and 50 µl of DEPC-treated water was added to the
   center of the membrane. It was allowed to stand at room temperature for
   2 min before centrifuging at 10,000 g for 1 min. Finally, the eluate
   was returned into the Hibind RNAMini column for another round of
   incubation at room temperature for 2 min, and centrifugation at
   10,000 g for 1 min was performed to obtain the purified RNA solution.
   To enrich the extracted mRNA, mRNA Capture Beads were employed.
   Following purification with these beads, the mRNA underwent
   fragmentation at elevated temperatures. The resulting fragmented mRNA
   served as a template for synthesizing the first strand of complementary
   DNA (cDNA) within a reverse transcription enzyme mixture. During the
   synthesis of the second strand of cDNA, processes such as end repair
   and A-tailing were also completed. Subsequently, adapters were ligated
   to the cDNA, and Hieff NGS^® DNA Selection Beads facilitated the
   purification of target fragments. The PCR amplification of the library
   followed, culminating in sequencing with the Illumina Novaseq X Plus.
   The sequencing output contained raw reads, which may have included
   adapters and low-quality bases that could have compromised downstream
   assembly and analysis. To ensure high-quality clean reads, fastp
   (version 0.18.0) [[56]17] was applied for further filtering; a
   filtering process was implemented that involved discarding any reads
   with adapter sequences, excluding those that contained over 10% unknown
   nucleotides (N), and removing low-quality reads characterized by more
   than 50% of their bases having a Q-value of 20 or lower. For mapping
   reads to the ribosomal RNA (rRNA) database, the short reads alignment
   tool Bowtie2 (version 2.2.8) [[57]18] was utilized, enabling the
   removal of rRNA-mapped reads. The resultant clean reads were then
   employed for assembly and gene abundance calculations. An index of the
   reference genome was constructed, and paired-end clean reads were
   mapped against this reference using HISAT2 (version 2.1.0) with default
   parameters [[58]19]. The mapped reads for each sample were assembled
   employing StringTie v1.3.1 through a reference-based approach [[59]20].
   For each transcription region, the expression abundance and variation
   were quantified by calculating the FPKM (fragments per kilobase of
   transcript per million mapped reads) value using RSEM software.
   Additionally, the mapped reads were utilized to compute a TPM
   (Transcripts Per Kilobase of exon model per Million mapped reads) value
   for further assessment of expression abundance and variability, also
   through RSEM. Differential expression analysis of RNAs was conducted
   using DESeq2 software to compare two distinct groups, with edgeR
   applied for sample comparison [[60]21]. Genes or transcripts meeting
   the criteria of a false discovery rate (FDR) below 0.05 and an absolute
   fold change ≥ 2 were classified as differentially expressed. Gene
   Ontology (GO) enrichment analysis identified significantly enriched GO
   terms among differentially expressed genes (DEGs) relative to the
   background of the genome. This mapping process involved associating all
   DEGs with GO terms from the GO database
   ([61]http://www.geneontology.org/), followed by a hypergeometric test
   to determine significant enrichment [[62]22]. Furthermore, genes
   typically engaged in interactions that facilitated specific biological
   functions. Pathway-based analysis enhanced the understanding of these
   functions. The KEGG database served as a primary public resource for
   pathway-related information [[63]23]. Pathway enrichment analysis
   discovered significantly enriched metabolic and signaling pathways
   within the DEGs when compared to the entire genome background.

Metabolomics studies

   The cell samples were ground using a pestle and mortar after being
   frozen in liquid nitrogen. A 100 mg aliquot was mixed with 1 mL of a
   cold solvent system consisting of methanol, acetonitrile, and water in
   a 2:2:1 volumetric ratio, followed by sonication at low temperature for
   30 min, performed twice. After centrifugation at 14,000 g for 20 min at
   4°C, the supernatant was isolated and subsequently dried using a vacuum
   centrifuge. A total volume of 100 µL of acetonitrile/water (1:1, v/v)
   was used to re-dissolve the samples for LC-MS analysis. Utilizing a
   UHPLC system (1290 Infinity LC, Agilent Technologies) paired with a
   quadrupole time-of-flight mass spectrometer (AB Sciex TripleTOF 6600,
   Shanghai), we conducted our analysis. The HILIC separation was executed
   on an ACQUITY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm particle
   size, Waters, Ireland). The mobile phase featured two components:
   component A was a blend of 25 mM ammonium acetate and an equal
   concentration of ammonium hydroxide in water, while component B
   comprised acetonitrile. Our gradient program began with 95% B and
   sustained this level for 0.5 min before gradually declining to 65% over
   a span of 6.5 min. Following this, the ratio was further decreased to
   40% for 1 min, maintaining stability for an additional minute before
   returning to 95% B in only 0.1 min, followed by a re-equilibration
   phase that lasted 3 min. We optimized the electrospray ionization (ESI)
   source parameters, adjusting Ion Source Gas 1 and Gas 2 to 60, the
   curtain gas to 30, and setting the source temperature at 600°C, with
   the IonSpray Voltage Floating (ISVF) configured to ± 5500 V. For the
   MS-only acquisition, we scanned the m/z range of 60 to 1000 Da,
   allowing for a TOF MS accumulation time of 0.20 s for each spectrum. In
   the auto MS/MS acquisition, the m/z range was modified to 25-1000 Da,
   with a product ion scan accumulation time set to 0.05 s per spectrum.
   We retained features that displayed over 50% nonzero measurements in
   any group, and compound identification was reliant on precise m/z
   values (within 10 ppm) as well as MS/MS spectra compared to an internal
   standard database. To increase the coverage of metabolites, we
   performed analyses in both positive and negative ion modes separately.
   The inclusion of quality control (QC) samples was essential for
   validating the reliability of our metabolomics study [[64]24]. We
   carried out principal component analysis (PCA) using the R language
   package gmodels (v2.18.1) [[65]25], where the denser clustering of QC
   samples indicated stronger reliability. Metabolites were prioritized
   based on their variable importance in projection (VIP) scores obtained
   from the (O)PLS model, establishing a threshold of VIP ≥ 1 for
   effective differentiation between the two groups. In addition, we
   executed a T-test for univariate analysis, successfully identifying
   differential metabolites at a significance threshold of FDR < 0.05.
   These metabolites were subsequently aligned with the KEGG database for
   annotation and enrichment analysis [[66]23].

Assessment of intracellular ATP levels and mitochondrial membrane potential

   To measure intracellular ATP levels and mitochondrial membrane
   potential, C. neoformans cells were initially cultured overnight in YPD
   medium at 30°C with shaking at 130 rpm. After this growth phase, the
   cells were washed twice with PBS. Intracellular ATP levels were
   measured using the BacTiter-Glo Microbial Cell Viability Assay kit
   (Promega, USA), and the signal was detected with a TECAN Infinite E
   Plex microplate reader. For evaluating mitochondrial membrane
   potential, the cells were treated with 50 nM Mito-Tracker Red CMXRos,
   which accumulates in mitochondria in a manner dependent on the
   mitochondrial membrane potential. This property allows it to serve as a
   probe for detecting changes in mitochondrial membrane potential, which
   can indicate apoptosis. The cells were incubated at 30°C for 1 h and
   subsequently washed twice with PBS. The fluorescence intensity of the
   sample, containing 5 × 10^7 cells, was analyzed using a TECAN Infinite
   E Plex microplate reader. All experiments were conducted with three
   biological replicates, and statistical significance was determined
   using a Student’s t-test.

Virulence assays

   For conducting virulence assays, we obtained 6-week-old female BALB/c
   mice from the Experimental Animal Center at Nantong University, located
   in Nantong, China. The WT strain and the taf1Δ mutant strain were
   cultivated overnight in YPD medium at 30°C. Following cultivation, the
   strains were washed with PBS and then resuspended in PBS at a
   concentration of 1 × 10^6 cells/ml. The inoculation process involved
   intranasal instillation of 50 µl of the cell suspension, resulting in
   an inoculum of 5 × 10^4 cells for each mouse. Each strain was tested
   using groups of 10 mice. After inoculation, the health status of the
   mice was assessed daily. CO[2] asphyxiation was utilized to euthanize
   mice that had reached the humane endpoint. This research adhered to the
   ethical guidelines regarding animal use set forth by Nantong
   University, ensuring that all mice were treated in a manner that
   minimized suffering. Survival analysis was conducted using Kaplan-Meier
   (K-M) curves and the log-rank (Mantel-Cox) test.

Results

TAF1-lacking mutant exhibits reduced virulence

   In our previous proteomic analyses of C. neoformans, we confirmed the
   expression of Taf1 [[67]11], a hypothetical protein encoded by the gene
   CNAG_04232 in the C. neoformans genome. Here we showed that the mutant
   taf1Δ displayed significantly impaired growth at 39°C (Fig. [68]1A).
   However, when cultured in media supplemented with L-DOPA
   (L-3,4-dihydroxyphenylalanine) under the same temperature conditions,
   the taf1Δ mutant exhibited normal melanin production (Fig. [69]1A). To
   explore the role of Taf1 in virulence-related phenotypes, we
   hypothesized that the absence of Taf1 would result in decreased
   pathogenicity in a murine model. To test this hypothesis, we
   intranasally inoculated mice with cells from both the WT strain and the
   taf1Δ mutant. By day 27 post-infection, all mice infected with the WT
   strain succumbed, while a significant proportion of mice infected with
   the taf1Δ mutant survived well beyond this timeframe (Fig. [70]1B).
   These findings suggest that Taf1 is essential for the virulence of C.
   neoformans.

Fig. 1.

   [71]Fig. 1
   [72]Open in a new tab

   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 10^5 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)

Mutant lacking TAF1 has decreased ATP levels and downregulated ATP-dependent
activities

   To further elucidate the molecular mechanisms associated with TAF1
   deletion, we performed a comprehensive analysis of the transcriptomic
   profiles of C. neoformans. RNA-Seq revealed that the absence of the
   TAF1 gene led to the upregulation of 204 genes and downregulation of
   908 genes (Fig. [73]2A and Supplementary Data 1). Subsequent GO
   analysis indicated that a significant proportion of these
   differentially expressed genes are involved in various biological
   processes, particularly in metabolic pathways, where most exhibited
   downregulation. Notably, a detailed classification of gene functions
   revealed a marked decrease in ATP-dependent activities, suggesting that
   Taf1 plays a critical role in maintaining energy homeostasis.
   Additional analysis of cellular components corroborated these findings,
   showing substantial downregulation of genes associated with cellular
   anatomical structures, which may have implications for cellular
   integrity and functionality (Fig. [74]3). Experimental assessments
   further confirmed a significant reduction in cellular ATP levels
   following the knockout of TAF1, as depicted in Fig. [75]4A.
   Interestingly, when we assessed the mitochondrial membrane potential of
   C. neoformans post-TAF1 deletion, we observed an unexpected increase in
   membrane potential in the knockout cells (Fig. [76]4B). This
   observation may indicate the activation of a compensatory mechanism in
   response to the energy deficit. Overall, these findings enhance our
   understanding of the intricate molecular mechanisms by which Taf1
   regulates cellular energy dynamics and metabolic processes in C.
   neoformans.

Fig. 2.

   [77]Fig. 2
   [78]Open in a new tab

   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.

   [79]Fig. 3
   [80]Open in a new tab

   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.

   Fig. 4
   [81]Open in a new tab

   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

Taf1 may impact fatty acid biosynthesis and degradation

   The deletion of TAF1 appears to affect fatty acid biosynthesis and
   degradation, as indicated by the KEGG pathway analysis of RNA-Seq data
   (Fig. [82]5). Our untargeted metabolomics approach further clarifies
   the metabolic changes associated with TAF1 deletion, which are
   summarized in Fig. [83]2B. In the negative ionization mode, we
   identified 19 distinct metabolite changes, with 8 metabolites showing
   increased levels and 11 displaying decreased levels (Fig. [84]2B and
   Supplementary Data 2). In the positive ionization mode, we detected 20
   individual alterations in metabolites, with 14 increasing and 6
   decreasing (Fig. [85]2B and Supplementary Data 2). Further KEGG pathway
   analysis corroborated these findings, revealing significant changes in
   pathways related to fatty acid biosynthesis and degradation, as well as
   alterations in oxidative phosphorylation and purine metabolism
   (Fig. [86]6). Overall, TAF1 may play a role in influencing fatty acid
   biosynthesis and degradation.

Fig. 5.

   [87]Fig. 5
   [88]Open in a new tab

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

Fig. 6.

   [89]Fig. 6
   [90]Open in a new tab

   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

Discussion

   In this study, we elucidate the role of Taf1, an uncharacterized
   protein, in regulating ATP levels and virulence in C. neoformans. Our
   findings demonstrate that the taf1Δ mutant exhibits impaired growth at
   39°C and reduced virulence in a murine model of cryptococcosis,
   underscoring the essential role of Taf1 in the infection process of C.
   neoformans.

   The ability to grow at temperatures between 37°C and 39°C is crucial
   for microbial infection in human hosts [[91]26]. Our results indicated
   that Taf1 was necessary for robust growth at 39°C, suggesting its
   involvement in the thermal adaptation required for virulence. Notably,
   at 30–37°C, the taf1Δ mutant exhibited growth comparable to that of the
   WT strain. This discrepancy raises an important question: why does the
   taf1Δ mutant display a growth defect only at 39°C and not at 30–37°C?
   We propose that this difference may be attributed to the distinct
   metabolic demands and stress responses elicited at higher temperatures.
   At 39°C, the taf1Δ mutant appears to struggle with thermal stress,
   which could compromise its ability to maintain essential cellular
   functions, including metabolic flexibility. In contrast, at 30–37°C,
   the mutant may be able to compensate for the loss of Taf1 through
   alternative pathways or mechanisms that are insufficient under the more
   stressful conditions at elevated temperatures. A deeper exploration of
   these temperature-dependent responses would enhance our interpretation
   of the results and provide insights into the mechanisms underlying the
   growth phenotype observed in the taf1Δ mutant.

   Transcriptomic analyses revealed that deletion of the TAF1 gene lead to
   the downregulation of genes associated with ATP-dependent activities.
   Our untargeted metabolomics further indicated alterations in oxidative
   phosphorylation and purine metabolism. Mitochondria serve as the
   primary sites for ATP synthesis, and this process is intricately linked
   to both oxidative phosphorylation and purine metabolism. While
   oxidative phosphorylation is the principal mechanism for ATP
   generation, purine metabolism is essential for ATP synthesis and
   various cellular functions. We hypothesize that Taf1 may regulate
   intracellular ATP levels by influencing these metabolic pathways, with
   the observed decline in ATP levels contributing to reduced
   ATP-dependent activities in the taf1Δ mutant. Additionally, our
   metabolomics analysis reveals significant alterations in fatty acid
   metabolism following TAF1 deletion. This finding connects Taf1 role in
   energy regulation to its impact on virulence, as fatty acids are
   critical not only for energy production but also as key components of
   cellular membranes and signaling molecules [[92]13, [93]16, [94]27].
   The interplay between ATP dynamics and fatty acid metabolism is
   particularly noteworthy; the reduction in ATP levels in the taf1Δ
   mutant correlates with impaired metabolic flexibility, which is vital
   for the pathogenic potential of C. neoformans. Future investigations
   should assess whether the taf1Δ mutant exhibits altered sensitivity to
   cell wall and membrane stressors. This metabolic flexibility is crucial
   for C. neoformans to adapt to the diverse and often hostile conditions
   encountered in the host environment, thereby enhancing its survival and
   virulence. The impaired growth at elevated temperatures observed in the
   taf1Δ mutant suggests a compromised ability to withstand physiological
   stresses during infection.

   Overall, these findings reinforce the notion that Taf1 is integral to
   the metabolic adaptability of C. neoformans, facilitating its
   navigation through complex environments and evasion of immune defenses.
   Furthermore, the differential expression profiles observed in the taf1Δ
   mutant provide a compelling foundation for future investigations into
   the specific metabolic pathways regulated by Taf1. Understanding these
   pathways could unveil new therapeutic targets aimed at disrupting
   energy regulation and virulence in C. neoformans. For instance,
   elucidating the precise mechanisms by which Taf1 influences fatty acid
   biosynthesis and degradation could lead to innovative strategies for
   enhancing antifungal efficacy and mitigating the impact of this
   pathogen.

Conclusion

   Our study provides evidence highlighting the role of Taf1 in regulating
   ATP levels, infection and fatty acid metabolism in C. neoformans. The
   complex interplay between Taf1, metabolic regulation, and pathogenicity
   underscores the necessity for further research to elucidate the
   molecular mechanisms underlying these processes. Gaining insight into
   these interactions is essential for developing novel therapeutic
   strategies aimed at effectively combating fungal infections and
   improving clinical outcomes for affected individuals.

Supplementary Information

   [95]Supplementary Material 1.^ (22.8KB, xlsx)
   [96]Supplementary Material 2.^ (762.8KB, xlsx)

Acknowledgements