With age comes resilience: how mitochondrial modulation drives age-associated fluconazole tolerance in Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans (Cn) is an opportunistic fungal microorganism that causes life-threatening meningoencephalitis. During the infection, the microbial population is heterogeneously composed of cells with varying generational ages, with older cells accumulating during chronic infections. This is attributed to their enhanced resistance to phagocytic killing and tolerance of antifungals like fluconazole (FLC). In this study, we investigated the role of ergosterol synthesis, ATP-binding cassette (ABC) transporters, and mitochondrial metabolism in the regulation of age-dependent FLC tolerance. We find that old Cn cells increase the production of ergosterol and exhibit upregulation of ABC transporters. Old cells also show transcriptional and phenotypic characteristics consistent with increased metabolic activity, leading to increased ATP production. This is accompanied by increased production of reactive oxygen species, which results in mitochondrial fragmentation. This study demonstrates that the metabolic changes occurring in the mitochondria of old cells drive the increase in ergosterol synthesis and the upregulation of ABC transporters, leading to FLC tolerance. IMPORTANCE Infections caused by Cryptococcus neoformans cause more than 180,000 deaths annually. Estimated 1-year mortality for patients receiving care ranges from 20% in developed countries to 70% in developing countries, suggesting that current treatments are inadequate. Some fungal cells can persist and replicate despite the usage of current antifungal regimens, leading to death or treatment failure. Aging in fungi is associated with enhanced tolerance against antifungals and resistance to killing by host cells. This study shows that age-dependent increase in mitochondrial reactive oxygen species drive changes in the regulation of membrane transporters and ergosterol synthesis, ultimately leading to the heightened tolerance against fluconazole in old C. neoformans cells. Understanding the underlying molecular mechanisms of this age-associated antifungal tolerance will enable more targeted antifungal therapies for cryptococcal infections.

In countries with limited resources where CME is most prevalent, fluconazole (FLC) is often the only available antifungal used for induction, consolidation, and long-term maintenance regimens (6).FLC works by inhibiting 14-alpha-demethylase, one of the key enzymes in ergosterol synthesis, thereby targeting an important component of the fungal plasma membrane (7).
One factor that contributes to the failure of FLC-based therapy is the development of drug tolerance or the ability of a subpopulation of cells to survive and grow in the presence of the drug at drug concentrations above the minimum inhibitory concen tration (MIC) (8)(9)(10)(11).As opposed to FLC resistance, FLC tolerance is not identified by standard MIC assays.FLC resistance in Cn can be mediated by changes in the ergosterol target and augmentation of drug efflux pumps, where mutations in ERG genes of the ergosterol biosynthesis pathway decrease the susceptibility to FLC (12,13), and overexpression of efflux pumps decreases intracellular drug concentration (14,15).The efflux of FLC is facilitated by the ATP-binding cassette (ABC) transporters, which require ATP to drive transport out of the cell (14).Most of the ATP synthesis occurs in mitochon dria, and biosynthesis of ergosterol starts from acetyl-coenzyme A (acetyl-CoA), a key mitochondrial intermediate.Furthermore, altered mitochondrial regulation has also been linked to FLC resistance in several pathogenic fungi (16)(17)(18)(19)(20).However, the connection among ergosterol regulation, ABC transporters, and mitochondrial metabolism and how it contributes to FLC tolerance is unknown.
Like other pathogenic fungi, Cn undergoes asymmetric cell division during replicative aging, where age is measured by the number of generations (21)(22)(23).During this process, proteins are unequally distributed between the mother and daughter cells, leading to age-dependent phenotypes (24).Older Cn cells that have undergone aging are more resistant to antifungals (15,25) and macrophage killing (26).As a result, older cells accumulate in cerebrospinal fluid both in a rat model and in humans during infection (27), supporting the concept that replicative aging conveys enhanced resilience within the host environment.
Cells of advanced generational age constitute the dominant subpopulation in vivo, which can be attributed to their enhanced FLC tolerance (27,28).However, the underlying mechanism behind the age-dependent FLC tolerance remains unknown.In this study, we explore the interplay between ergosterol regulation, mitochondrial metabolism, and ABC transporters in mediating age-dependent FLC tolerance in old Cn cells.We show that old Cn cells exhibit altered metabolic activity, leading to increased ATP production that fuels ABC transporters, as well as upregulation of ergosterol synthesis.We further demonstrate that the increased mitochondrial burden in old cells leads to reactive oxygen species (ROS)-mediated mitochondrial signaling, resulting in alteration in ergosterol and ABC transporter dynamics that contribute to age-asso ciated FLC tolerance.These results offer insight into the molecular dynamics govern ing age-associated mitochondrial signaling with its repercussions on drug tolerance, providing a foundation for more targeted therapeutic interventions.

Transcriptomic impact of replicative aging in Cn
To investigate the effects of replicative aging on gene expression in Cn, we performed RNA-seq analyses on young and old Cn cells.Aging resulted in the differential regulation of 1,266 genes (adjusted P-value ≤ 0.05), including 725 upregulated and 541 downregu lated genes (Fig. 1A).Heat map visualization of RNA-seq data and Pearson's Correlation Analysis of the biological replicates indicated the reliability of our RNA-seq results (Fig. S1).Further exploration using gene ontology (GO) enrichment analysis unveiled an upregulation of genes associated with 27 biological processes (BPs; Fig. 1B).Additionally, genes related to 19 molecular functions (MFs) were upregulated in old cells (Fig. 1C).Conversely, aging correlated with the downregulation of genes linked to 34 biological processes and 23 molecular functions (Fig. S2).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also indica ted that some important pathways, such as peroxisome, fatty acid degradation, and steroid biosynthesis, were enriched in old Cn cells (Fig. 1D).One of the key functions of peroxisomes is the β-oxidation of fatty acids (29), which produce acetyl-CoA that can serve as a substrate for ATP production through the tricarboxylic acid (TCA) cycle.Furthermore, acetyl-CoA is the central metabolite for the biosynthesis of ergosterol, the most abundant sterol in fungal cell membranes.Because ATP-powered ABC transporters and ergosterol biosynthesis are both implicated in mediating FLC tolerance, we looked more closely at how aging affects gene expression of these aspects of metabolism.As indicated by the KEGG analysis, various genes that encode enzymes in sterol biosynthesis and fatty acid oxidation were upregulated in old Cn cells (Table 1).Genes associated with respiratory functions, such as ones encoding cytochrome C oxidases II and III, apocytochrome C, and nicotinamide adenine dinucleotide (NADH) dehydrogenases, and genes encoding various mitochondrial functions also exhibited significant upregulation in aged cells.Altogether, these results from the transcriptomic analysis of young and old Cn cells indicate that aging affects various metabolic pathways that are linked with FLC tolerance.

Integration of transcriptomic and phenotypic changes in old Cn cells
The upregulation of genes that encode metabolic pathways linked with FLC tolerance in aged Cn cells prompted an investigation into whether these changes align with phenotypic characteristics.We first assessed if the enhanced expression of genes associated with respiration in aged cells corresponds to increased ATP production.Confirming our hypothesis, old Cn cells exhibi ted elevated cellular ATP levels compared to their younger counterparts (Fig. 2A).This observation was corroborated by heightened extracellular reduction  of XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2h-tetrazo lium hydroxide}, a result of NADH produced in the mitochondria (Fig. S3).Quantification of mitochondrial mass (MM) using Mitotracker Green FM fluorescence also revealed a higher MM in old Cn cells (Fig. 2B), indicating heightened mitochondrial respiration in old Cn cells.Next, we explored the role of neutral lipids as potential energy sources via β-oxidation through the TCA cycle within the mitochondria.These lipids are stored in lipid droplets (LDs), which can be mobilized to the vacuole for energy (30,31).Quantifying LDs using BODIPY 494/503 fluorescence demonstrated an increase in old Cn cells compared to their younger counterparts (Fig. 2C).Next, we performed confocal microscopy to visualize the LDs and probe their potential localization to the vacuole (Fig. 2D).We observed that the number of LDs per cell and the percent of LDs localized to the vacuole were higher in aged cells (Fig. 2E).This mobilization of LDs to the vacuole also supported the augmentation of acetyl-CoA pools through β-oxidation, as evidenced by the significant increase in acetyl-CoA levels in old Cn cells (Fig. 2F).
To gain insights into the specific metabolic pathways contributing to the observed differences in energy production, we compared the metabolic flux between young and old Cn cells using the Seahorse XF Analyzer (Fig. 2G and H).Basal extracellular acidifica tion rate (ECAR) and oxygen consumption rate (OCR) were similar, indicating comparable levels of glycolysis, TCA cycle, and electron transport chain (ETC) activity under glucosedepleted conditions.However, following glucose injection, glycolysis and respiration increased more efficiently in old than in young Cn cells, as indicated by a higher rise in ECAR and OCR.When challenged with inhibitors of complex I and III (rotenone/antimycin A [Rot/AA]), old cells maintained higher glycolytic and TCA cycle activity in the absence of the ETC.Furthermore, adding glycolysis inhibitor 2-deoxyglucose (2-DG) led to a similar moderate decrease in OCR in both groups, indicating a reduction in baseline levels.

Mitochondrial morphology dynamics in old Cn cells
The dynamic reorganization of the mitochondrial network, characterized by fission and fusion events, plays a pivotal role in responding to cellular energy demands (32,33).We, therefore, investigated the impact of increased energy production in aged Cn cells on their mitochondrial morphology.Utilizing the mitochondrionspecific fluorescent dye, MitoTracker Green FM, and deconvolution fluorescence microscopy, we observed distinct differences in mitochondrial morphologies between young and old Cn cells, where young cells displayed diffuse mitochondrial networks while old cells exhibited highly fragmented mitochondria (Fig. 3A).
Given the distinct mitochondrial morphology in aged Cn cells, we explored the role of dynamin-related proteins (DRPs) in mediating mitochondrial fusion and fission (34).Genetic manipulations, including single-deletion strains for each of the fission genes (ΔFis1, ΔMdv1, and ΔDnm1) and overexpression of the fusion gene (Fzo1-OE), revealed that these alterations influenced mitochondrial fragmentation in old cells (Fig. 3B).Notably, defects in fission and increased fusion led to the loss of mitochondrial fragmentation, emphasizing the role of DRPs in age-associated changes.
Considering the observed increase in metabolic activity in old Cn cells, we hypothe sized that the mitochondrial burden might contribute to the observed fragmentation.To test this hypothesis, we investigated whether mitochondrial oxidative stress, a byprod uct of increased ATP production (35), played a role in mitochondrial fragmentation.Treatment with MitoTEMPO, a mitochondrially targeted antioxidant, resulted in similar mitochondrial morphologies in both young and old cells (Fig. 3C), suggesting that mitochondrial superoxide facilitates age-associated mitochondrial fragmentation in old cells.

Age-associated changes in ROS levels, superoxide dismutase response, and antioxidant defense mechanisms
The increased mitochondrial burden resulting from ATP production in aged Cn cells prompted a deeper exploration of the mitochondrial stress response.First, we assessed cellular reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels, indicative of oxidative stress resulting from ATP production through oxidative phosphorylation.Old Cn cells exhibited elevated H 2 DCFDA and MitoSox Red fluorescence, signifying increased cROS (Fig. 4A) and mROS (Fig. 4B), respectively.Furthermore, we observed a higher mitochondrial calcium level in old Cn cells, as indicated by increased Fluo-4 AM fluorescence (Fig. 4C).
A crucial mechanism in response to elevated ROS is the action of superoxide dismutase (SOD), which converts ROS to harmless water and free oxygen (36).In Cn, Sod1 and Sod2 regulate cROS and mROS, respectively (37,38).RT-qPCR analysis revealed the upregulation of SOD1 and downregulation of SOD2 in old Cn cells (Fig. 4D).Intriguingly, the intracellular localization of Sod2-GFP in old cells showed dispersed cytoplasmic distribution, contrasting with the predominantly mitochondrial localization in young Cn cells (Fig. 4E).
To reconcile the downregulation of SOD2 as well as cytoplasmic localization of Sod2-GFP despite the high mROS in old Cn cells, we explored the regulation of other components of the cellular antioxidant defense system.Our findings revealed an upregulation of genes encoding copper-detoxifying metallothionein 1 (MTN1), thioredoxin reductase (TRR1), catalase 4 (CAT4), mitochondrial cytochrome c peroxidase (CCP1), thiol peroxidase 1 and 3 (TSA1 and TSA3), and glutathione peroxidase 1 (GPX1) in old Cn cells (Fig. 4F).Expression levels of other genes encoding other components of the cellular antioxidant defense system were unchanged (Fig. S4).

Role of ABC transporters and ergosterol dynamics in age-associated FLC tolerance
The development of FLC resistance in Cn has been linked to enhanced expression and activity of efflux pumps, resulting in decreased intracellular drug concentrations (14,15).Given their involvement in drug efflux, we investigated the potential contribution of ABC transporters to age-associated FLC tolerance in old Cn cells.Quantification of the mRNA expression of key ABC transporters revealed a significant upregulation of AFR1, AFR2, AFR3, and MDR1 in old cells (Fig. 5A).All of these genes, with the exception of AFR3, were confirmed to be also upregulated in our RNA-seq analysis.To further confirm the potential role of the ABC transporters in age-associated FLC tolerance, we assessed their efflux activity using fluorescent probes Nile red (NR) and rhodamine 6G (R6G) (39)(40)(41).The results demonstrated heightened efflux of both NR and R6G in old Cn cells (Fig. 5B  and C), indicating increased activity of ABC transporters in aged cells.
Considering that changes in ergosterol levels also contribute to FLC resistance, we explored ergosterol dynamics in young and old Cn cells.First, we performed gas chromatography-mass spectrometry (GC-MS) to measure the total ergosterol levels of young and old (Fig. 5D).GC-MS analysis demonstrated higher ergosterol levels in old Cn cells compared to young cells (Fig. 5E).This result was further supported by filipin staining, a fluorescent dye that binds to membrane sterols (42) (Fig. 5F).To understand the molecular basis of altered ergosterol levels, we examined the mRNA expression of genes involved in the ergosterol synthesis pathway.Out of 23 genes, old cells exhibited significant upregulation of 18 genes (log 2 FC old/young ≥ 1) and downregulation of 1 gene (log 2 FC old/young ≤ −1; Fig. 5G), emphasizing the regulatory changes in ergosterol synthesis during aging.

Mitochondrial signaling dynamics contributing to age-associated FLC tolerance
Mitochondria, beyond their canonical role in ATP production, orchestrate cellular responses, engaging in intricate crosstalk with various organelles and cellular path ways (18,(43)(44)(45).Considering the distinctive mitochondrial characteristics exhibited by aged Cn cells, we tested the possibility that the mitochondrial stress exhibited by old cells leads to downstream cellular signaling.Drawing insights from previous studies demonstrating the influence of mitochondrial signaling on drug resistance in fungal species (16)(17)(18)(19)(20), we hypothesized that the observed mitochondrial stress in aged Cn cells might trigger downstream signaling pathways contributing to their FLC tolerance.
To test this hypothesis, we first probed the impact of antioxidants on FLC tolerance by examining the specific effects of ascorbic acid (AA) and glutathione (GSH).Both young and old cells treated with antioxidants exhibited diminished cROS levels compared to untreated controls (Fig. S5A).While these antioxidants significantly reduced FLC killing in young cells, they did not affect FLC killing in aged cells (Fig. 6A).Further exploration involved targeted scavenging of mitochondrial superoxide using MitoTEMPO (46).While MitoTEMPO generally attenuated FLC killing of young cells (Fig. 6B), the response in aged cells proved to be more complex.Contrary to our expectations, aged cells exhibited increased FLC killing in the presence of MitoTEMPO, surpassing the effect observed in young cells.We also confirmed that young and old cells treated with MitoTEMPO displayed diminished mROS levels compared to untreated controls (Fig. S5B).
These results prompted a deeper inquiry into how mitochondrial superoxide influences age-associated FLC tolerance.First, we tested the possibility that MitoTEMPO treatment alters the metabolic upregulation that we observed in old cells.However, Seahorse analysis revealed that MitoTEMPO does not affect the metabolic flux that was previously observed in young and old cells (Fig. S6A and B).Furthermore, MitoTEMPO did not affect the age-associated increase in intracellular ATP levels (Fig. S6C).Next, we probed how the addition of MitoTEMPO influences the changes in ABC transporter expression and increased efflux seen in old cells.When the cells underwent replicative aging in the presence of MitoTEMPO, old cells showed a slight downregulation of AFR1 and AFR2 compared to young cells, while there were no significant differences in AFR3 and MDR1 expression (Fig. 6C).These transcriptional changes were consistent with efflux activity, as young cells demonstrated higher efflux activity measured by fluorescent probes NR and R6G (Fig. 6D and E).Because Afr1 is the dominant membrane transporter in Cn (14,15,47), we also asked if Afr1 is sufficient to drive age-associated FLC tolerance.Despite the importance of Afr1 in azole resistance, aged Afr1Δ cells still demonstrated significant age-associated FLC tolerance, and the presence of MitoTEMPO resulted in increased FLC killing in these mutants (Fig. S5C).These results suggest that the observed age-associated FLC tolerance in old Cn cells is mediated by more than just the upregula tion of Afr1.
We also examined whether mROS influences age-associated changes in ergosterol regulation.Using GC-MS and filipin staining, we observed no significant difference in ergosterol levels in young and old cells grown in the presence of MitoTEMPO (Fig. 6F,  G and H).Upon further interrogation, we found no significant differential expression of genes involved in the ergosterol synthesis pathway between young and old cells grown in the presence of MitoTEMPO (Fig. 6I).Taken together, the loss in age-dependent changes in ABC transporters and ergosterol synthesis emphasizes the importance of mROS in mediating these changes to drive age-associated FLC tolerance in Cn.

DISCUSSION
This is the first study to interrogate the mechanisms by which replicative aging confers increased FLC tolerance in Cn.Ten-generation-old Cn cells demonstrated widespread transcriptional changes that encompass various aspects of metabolism.These cells displayed increased mitochondrial activity that resulted in increased ATP production, which fuels ABC transporters.These changes are accompanied by the upregulation of ergosterol synthesis and ABC transporters, which are dependent on the mROS signaling resulting from increased mitochondrial stress.These findings constitute the mechanistic basis that underlies the age-associated FLC tolerance in old Cn cells (Fig. 7).Using GO and KEGG enrichment analyses, we were able to classify differentially expressed genes according to their molecular function, biological processes, and biological pathways.We found that various genes that encode fatty acid oxidation enzymes and genes associated with respiratory and mitochondrial functions exhibited significant upregulation in aged cells.β-Oxidation of fatty acids produces acetyl-CoA, a substrate for ATP production through the mitochondria-driven TCA cycle and a central metabolite for ergosterol biosynthesis.Because ATP-powered ABC transporters and ergosterol biosynthesis are both implicated in mediating FLC tolerance, we examined how aging in Cn induces changes in mitochondrial metabolism and how these changes affect ABC transporters and ergosterol synthesis to drive FLC tolerance.
To complement the results from our transcriptomic analyses, we looked to iden tify phenotypic characteristics that were consistent with the upregulation of cellular respiration through fatty acid oxidation.The ability of Cn cells to produce energy through respiration is crucial for cryptococcal virulence, in vivo resilience, and antifungal resistance (48,49).We found that older Cn cells produce higher intracellular ATP levels.This result was also confirmed by the XTT assay, a viability staining method that can serve as a marker for mitochondrial activity based on the reduction of XTT by mitochon drial dehydrogenase.Lastly, older Cn cells demonstrated higher mitochondrial mass compared to younger cells, further confirming characteristics consistent with their high ATP levels.
The majority of ATP is synthesized in the mitochondria during respiration and fatty acid oxidation.Neutral lipids are stored in LDs, and hydrolysis of LDs provides an extra source of energy through fatty acid oxidation (50).LDs communicate via direct contact with other cellular organelles, and our data demonstrate that Cn LDs localize to the vacuole regardless of the cells' replicative age.In yeast, the vacuole functions as the major catabolic organelle and interacts with LDs to regulate lipid homeostasis (30,31,50).Notably, older cells exhibited a higher amount of total intracellular LDs, as well as a larger number of LDs per cell and higher percentage of LDs localized to the vacuole compared to young cells.Furthermore, we observed an increase in intracellular acetyl-CoA levels in the old cells, indicating higher fatty acid oxidation.Altogether, these results indicate that older Cn cells have a higher cellular supply of energy sources that can respond to cellular metabolic demands.
The Seahorse analysis also revealed some notable changes in metabolic flux in old Cn cells.First, the old cells demonstrated a higher maximal level of glycolysis and respiration, as well as a higher rate of response to glucose.These are supported by the higher OCR/ECAR maximum in old cells and the higher increase in OCR/ECAR levels immediately following glucose injection.These differences indicate that older cells sense and transport glucose faster than young cells.Additionally, older cells may have a higher metabolic turnover and demand, leading them to respond to nutrients faster and at a higher maximum capacity than young cells.Another notable difference was that old cells also showed significantly higher glycolysis following the inhibition of mitochondrial oxidative phosphorylation by Rot/AA.Because residual glycolysis at this stage is dependent on the NAD+ levels in Cn (51), it could be that a larger NAD+ pool in old Cn cells drives glycolysis to meet the increased energy demand.
To maintain proper functionality, the mitochondrial network undergoes constant dynamic reorganization that leads to changes in mitochondrial morphologies (34).During healthy growth, mitochondria exhibit a diffuse morphology.However, under various cellular stresses, mitochondria can undergo fusion and fission, leading to tubular and fragmented mitochondrial morphologies, respectively (52).These morphological changes are facilitated by DRPs, where fission proteins Fis1, Mdv1, and Dmn1 medi ate mitochondrial fragmentation (34).We observed progressive fragmentation of the mitochondria upon replicative aging, where young cells displayed diffuse mitochondrial networks while old cells exhibited highly fragmented mitochondria.In contrast, we did not observe age-associated mitochondrial fragmentation in aged in fissiondefective strains (∆Dnm1, ∆Mdv1, and ∆Fis1), indicating that all three fission proteins are necessary to mediate mitochondrial fragmentation seen in aging.
Studies have shown that an increase in oxidative stress during replicative aging results in mitochondrial network fragmentation (53,54).This increase in oxidative stress originates from ROS, an unavoidable byproduct of respiratory chain function during ATP synthesis in a eukaryotic cell.Indeed, we observed higher cROS as well as higher mROS in older Cn cells than in younger cells.Quenching the mROS by adding MitoTEMPO during the aging process resulted in similar mitochondrial morphologies between young and old cells, indicating that the increased mROS production during replicative aging contributes to the mitochondrial fragmentation observed in old Cn cells.This was further supported by the high mitochondrial calcium levels, as mitochondrial calcium overload can also be a form of mitochondrial stress that induces mitochondrial fragmentation (55).
SOD serves as an important antioxidant defense by catalyzing the breakdown of ROS to minimize the cellular damage caused by the generation of superoxide during respiration (36).While we observed the upregulation of SOD1 in response to higher cROS in old Cn cells, we also detected downregulation of SOD2 despite the elevated mROS.Furthermore, we saw cytoplasmic localization of Sod2 in older cells, contrasted with mitochondrial localization in young cells.We also detected a widespread upregulation of other antioxidant response proteins in old cells.Some of these proteins, such as Mtn1, Trr1, Ccp1, and Tsa3, are known to localize to the mitochondria (56)(57)(58).The presence of this redundancy in the mitochondrial antioxidant defense system might explain the downregulation of SOD2 and cytoplasmic localization of Sod2 seen in old Cn cells.Notably, we also observed an upregulation of Cat4 and Gpx1, which both catalyzes the further detoxification of hydrogen peroxide, produced by SOD from superoxide, into water and oxygen.
The efflux pumps that are associated with FLC resistance in Cn belong to the ABC transporter family, which requires an adequate supply of ATP for FLC efflux (14,15,47,59,60).Afr1 is the dominant membrane transporter in Cn, while Afr2, Afr3, and Mdr1 have also been shown to play a role in azole resistance (14,15,47).Consistent with our previous study demonstrating that older Cn cells are more resistant to FLC (15), we saw significant upregulation of all four ABC transporters in these cells.Interestingly, we observed the lowest level of fold change in Afr1 out of the four ABC transporters despite the relative significance of Afr1 in mediating azole resistance.Furthermore, although the absence of Afr1 increased the susceptibility of Cn cells to FLC, old ΔAfr1 cells still demonstrated age-associated FLC tolerance.These results suggest that even though Afr1 is the dominant membrane transporter in Cn, it is not solely responsible for driving the age-associated FLC tolerance.Using fluorescent probes NR and R6G to measure efflux activity, we validated the upregulation of ABC transporters in older cells.This difference in NR and R6G efflux activities is also consistent with the higher ATP levels seen in old cells, which serve as fuel to drive these cellular efflux pumps.
Disruptions to the ergosterol metabolic pathway have been implicated in both diminished effectiveness of azole compounds (12,13).Using GC-MS, we demonstrated that old Cn cells contain higher ergosterol levels compared to young cells.Consistent with this finding, we also observed increased filipin III fluorescence and significant upregulation of 18 out of 23 genes involved in the ergosterol synthesis pathway in old cells.Notably, ERG11, encoding the enzyme that is directly targeted by FLC (7), and ERG6, which is crucial for Cn virulence by affecting membrane integrity and dynamics (61), were upregulated.Ergosterol biosynthesis starts with the condensation of two acetyl-CoA molecules (62), and ergosterol can be stored in LDs in the form of steryl ester (63).Interestingly, we also found higher levels of acetyl-CoA and LDs in old cells, further supporting our results that old cells upregulate ergosterol synthesis.
To examine the potential role of increased ROS in mediating the age-associated FLC tolerance in old Cn cells, we tested the FLC tolerance of old cells that have undergone replicative aging in the presence of various antioxidants.General antioxidants like AA and GSH significantly reduced FLC susceptibility in young cells but had no effect in aged cells.This observation is consistent with previous studies that have shown that ROS contributes to FLC-mediated growth inhibition, which can be rescued by using these antioxidants (64)(65)(66).Interestingly, aged Cn cells in the presence of MitoTEMPO exhibited increased susceptibility to FLC, suggesting that oxidative stress response, which mediates age-associated FLC resistance in old cells, might be specific to mROS.
Old cells that have undergone replicative aging in the presence of MitoTEMPO, compared to their younger counterparts, displayed downregulation of AFR1 and AFR2 and no significant differential regulation of AFR3 and MDR1.These transcriptional changes were also supported by NR and R6G studies demonstrating the decreased efflux activity of these cells.Because old cells treated with MitoTEMPO still display age-associated in ATP levels and metabolic flux (Fig. S6), the decreased efflux activity of these cells is more likely due to the transcriptional changes in efflux pumps than the changes in ATP that drive the efflux pumps.Furthermore, these MitoTEMPO-treated old Cn cells exhibited no difference in ergosterol levels and no longer displayed widespread significant upregulation of ergosterol synthesis genes.These distinct changes in ABC transporter and ergosterol phenotypes brought on by scavenging superoxide confirmed that mROS plays a pivotal role in mediating the observed age-associated characteristics that contribute to the resistance of old Cn cells against FLC.
Several studies have highlighted the role of mitochondria-driven signaling in mediating FLC resistance in pathogenic fungi.In Candida glabrata, the loss of mito chondrial function is associated with the acquisition of azole resistance through the upregulation of genes encoding ABC transporters (17,67).In Saccharomyces cerevisiae and Aspergillus fumigatus, mitochondrial dysfunction arising from alteration of cofilin residues has been shown to trigger retrograde signaling, resulting in the upregulation of ABC transporter genes and activation of fatty acid β-oxidation to increase acetyl-CoA and ATP levels (16,68).In Cn, Mar1 has been shown to mediate FLC tolerance by altering cellular mitochondrial metabolism (69).Additionally, transcriptomic analysis of Cn during exposure to oxidative stress revealed that there is an induction of an antifungal drug resistance response upon the treatment of Cn with hydrogen peroxide (70).Furthermore, loss of Mig1, a zincfinger protein that regulates mitochondrial respiration, ROS, and iron metabolism, results in increased susceptibility to FLC (19).These studies support our conclusions that increased mROS promotes FLC tolerance in old Cn cells.This is the first study that highlights the mechanism that underlies age-associated FLC tolerance in Cn cells of advanced replicative age.Replicative aging results in a heterogeneous cell population composed of cells with varying generational ages.Cells of advanced generational age accumulate during in vivo Cn and C. glabrata infection, indicating that they are selected by the host environment (27,28).We demonstrate that aging in Cn cells modulates mitochondrial metabolism and promotes FLC tolerance, ultimately contributing to their resilience.Genetic antifungal resistance is uncommon among Cn (10,11) and does not explain the poor clinical outcomes that are observed with cryptococcal meningitis (71).Our findings may shed light on why standard antifungal resistance testing performed with in vitro-grown Cn populations does not predict treatment failure.Understanding the non-genetic age-dependent heterogeneity in fungal populations not only expands our comprehension of drug tolerance but also greatly advances our endeavors in identifying novel therapeutic targets.

Strains and media
Cn strains were kept on yeast peptone dextrose (YPD) agar plates.Synthetic medium (SM) (72) was used for the cultivation of yeast cells.Ten millimolar ascorbic acid, 10 mM glutathione, and 50 µM MitoTEMPO were added to the media when needed.All the strains used in this study are listed in Table S1.

Isolation of young and old Cn cells
The isolation of young (0-3 generations) and old (10 generations) Cn cells was performed as previously described (73).

RNA-seq analysis
RNA was extracted using the RNAeasy Plus kit, following the manufacturer's guidelines.RNA-seq was performed by Novogene Co. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads and converted to cDNA.Libraries were clustered and sequenced on an Illumina NovaSeq 6000 platform, which utilizes a paired-end 150 bp sequencing strategy according to the manufacturer's instructions.Raw data (raw reads) of fastq format were first processed through in-house perl scripts, where clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N, and low-quality reads from raw data.At the same time, Q20, Q30, and GC content of the clean data were calculated.All the downstream analy ses were based on clean data with high quality.Differential expression analysis was performed using the DESeq2Rpackage (1.20.0).The resulting P-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate.Genes with an adjusted P-value ≤ 0.05 and |log 2 fold change| ≥ 1 were assigned as differentially expressed.GO enrichment analysis was performed by running differen tially expressed genes through FungiDB, selecting for BP and MF with a P-value cutoff of 0.05.KEGG analysis was performed by using clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways.Volcano plots and heatmaps were generated by Novogene Co.

Measurement of cellular ATP levels
Cellular ATP levels were quantified using the ATP Bioluminescent Assay Kit as previously described (72).The assay was performed in triplicate.

XTT assay
Cellular XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] levels were quantified using XTT Assay Kit following the manufacturer's guidelines.Briefly, 5 × 10 6 young and old cells were washed three times with SM and resuspended in 500 µL SM.For each reaction, 150 µL activated XTT solution was added and was incubated at 37°C for 3 hours protected from light.Following incubation, cells were pelleted, and the supernatant fraction was measured for absorbance at 475 nm with the spectrophotometer.Heat-killed cells (5 min incubation at 65°C) were used as negative controls.The assay was performed in triplicate.

Measurement of cellular acetyl-CoA levels
Ten million cells were mixed with 400 µL assay buffer and vortexed for 15 min in the presence of sterile acid-washed glass beads to initiate lysis.Following lysis, samples were retrieved by removing the cellular debris and beads by centrifugation.Fifty microliter samples were loaded in black 96-well plates, and 10 µL of acetyl-CoA quencher was added to each sample and incubated at room temperature for 5 min.Then, 2 µL of quench remover was added and incubated for an additional 5 min.Samples were mixed with 2 µL acetyl-CoA substrate mix, 1 µL conversion enzyme, 5 µL acetyl-CoA enzyme mix, and 2 µL fluorescent probe.The reaction was incubated for 10 min at 37°C, and fluorescence was read in a fluorescent plate reader with λex = 535 nm/λem = 587 nm.Acetyl-CoA standard solution was used in serial dilution to generate a standard curve, which was used to interpolate the cellular acetyl-CoA levels.The assay was performed in triplicate.

Quantification of cellular phenotypes using fluorescent dyes
Young and old cells (10 6 ) were stained and protected from light with the following dyes with the respective concentrations and incubation times/temperature: 10 µM H 2 DCFDA for 30 min at 37°C, 500 nM MitoSOX Red for 15 min at 37°C, 200 nM MitoTracker Green FM for 30 min at 37°C, 5 µM BODIPY 493/503 for 30 min at 24°C, and 5 µg/mL filipin III for 5 min at 24°C.After staining, the cells were washed three times and resuspended in phosphatebuffered saline (PBS), and 200 µL of washed stained cells were loaded in black 96-well plates.Fluorescence was read in a fluorescent plate reader with the following wavelengths: λex = 495 nm/λem = 525 nm for H 2 DCFDA, λex = 510 nm/λem = 580 nm for MitoSOX Red, λex = 490 nm/λem = 516 nm for MitoTracker Green FM, λex = 491 nm/λem = 516 nm for BODIPY 493/503, and λex = 360 nm/λem = 470 nm for filipin III.Fluorescence was measured in relative fluorescence intensity (RFI) by subtracting the RFI values between the stained and non-stained cells.Experiments were performed in biological triplicate.

Microscopy
To visualize the mitochondria, fungal cells (10 6 ) were stained with 500 nM MitoTracker Green FMand 500 nM MitoTracker Red FM for 30 min at 37°C.After staining, cells were fixed and imaged at 100× using a deconvolution microscope (Zeiss Axiovert 200M), using the FITC (for MitoTracker Green FM) and Texas Red (for MitoTracker Red FM) as previously described (72).Each experiment was repeated at least once on different days under the same conditions.
To visualize the vacuole and lipid droplets, fungal cells (10 6 ) were first stained with 16 µM FM 4-64 for 30 min at 30°C.After staining, cells were washed once with YPD, resuspended in fresh YPD, and incubated for an additional 2 hours at 30°C.Then, cells were washed once with YPD and stained with 5 µM BODIPY 493/504 for 30 min at RT. Lastly, cells were washed twice, resuspended in a 1:1 mixture of PBS and antifade mounting medium, and mounted on a slide using a poly-L-lysine treated coverslip.Cells were imaged at 100C using a confocal microscope (Zeiss LSM 980) using the FITC (for BODIPY 493/504) and Texas Red (for FM 4-64).Finally, images were processed using ZEN 3.6 (blue edition) to adjust contrast and brightness.

Seahorse XF analysis
A glycolytic rate assay was performed on a Seahorse Biosciences XFe96 extracellular flux analyzer for OCR and ECAR as previously described (51) with minor modifications.Briefly, cartridges were hydrated overnight in Agilent Seahorse XF calibrant.One hundred eighty microliter of young and old cells (2 × 10 6 cells/mL) was loaded into each well.Assay solutions were injected to a final concentration of 20 mM glucose, 3.5 µM Rot/AA, and 100 mM 2-DG.Cells and solutions were prepared on Seahorse XF Base Medium supplemented with 2 mM L-glutamine and 5 mM HEPES, pH 7.4.Each condition was measured with at least 15 replicates.

RT-qPCR analysis
RNA extraction, cDNA conversion, and qPCR were performed following the manufactur ers' guidelines as previously described (15).The oligonucleotides used in this study are described in Table S2.The assay was performed in biological triplicate.

NR and R6G efflux assays
NR and R6G assays were performed to analyze the efflux activities of the membrane transporters as previously described (15,72).The assays were performed in biological triplicate.

FLC killing assay
FLC killing assay was performed as previously described (15).Percent killing was analyzed by the following formula: % Killing = CFUs from well without FLC -CFUs from well with FLC CFUs from well without FLC x 100 The assay was performed in biological triplicate.

Ergosterol quantification by GC-MS
Lipid extraction and GC-MS were conducted as previously described (62).Briefly, cell pellets with 1 × 10 8 cells were used for lipid extraction.The dried total samples were resuspended in 100 µL chloroform added to 100 µL of N,Obis(trimethylsilyl)trifluoroace tamide (BSTFA) reagent and incubated at 70°C for 1 hour prior to GC-MS analysis.The retention time and mass spectral patterns of a sterol standard were used as references for lipid analysis.Cholesterol was added as an internal standard for these analyses prior to lipid extraction.The assays were performed in biological triplicate.

Statistics
All statistical analysis was performed using Prism 10.2.1.The individual statistical tests performed in each assay are described in the figure legends.

FIG 1
FIG 1 Replicative aging results in transcriptomic changes related to metabolic processes in C. neoformans.RNA-seq of young (1-3 generations) and old (10 generations) Cn cells was performed.(A) Volcano plot of genes that are differentially regulated in old and young cells.Genes that are upregulated in old vs young (log 2 FC ≥ 1, adjusted P ≤ 0.05) are indicated by pink points, while genes that are downregulated in old vs young (log 2 FC ≤ −1, adjusted P ≤ 0.05) are indicated (Continued on next page)

FIG 1 (
FIG 1 (Continued) by green points.Genes that do not meet these criteria are shown in blue.GO terms of differentially expressed genes show (B) MF and (C) BP that are upregulated in old Cn cells.Presented are the top 20 GO terms based on the lowest over-represented P-values.(D) Kyoto Encyclopedia of Genes and Genomes analysis of differentially expressed genes shows molecular pathways that are upregulated in old Cn cells.

FIG 2 (
FIG 2 (Continued) was performed to determine the P-value (*P < 0.05 and ***P < 0.001).(D) Vacuolar structure and LDs of young and old Cn cells were visualized by confocal microscopy after staining with FM 4-64 and BODIPY 493/503, and shown are the representative images.Scale bars in white = 5 µm.(E) The number of LDs per cell and percentage of LDs colocalized by vacuole were determined from 50 independent young and old cells, and Student's t test was performed to determine the P-value (****P < 0.0001).(F) Acetyl CoA levels of young and old cells were quantified using acetyl-coenzyme a fluorescent assay.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (*P < 0.05).(G) Extracellular acidification rate (ECAR) and (H) oxygen consumption rate (OCR) profiles of young and old cells were generated from the Seahorse XF glycolysis stress test following injection of glucose (GLC), rotenone/antimycin A (Rot/AA), and 2-deoxyglucose (2-DG).Plotted values signify the mean ± SEM of 15 replicates.

FIG 3
FIG 3 Dynamin-related proteins (DRPs) and mitochondrial reactive oxygen species govern age-associated mitochondrial fragmentation in old Cn cells.(A) Mitochondrial morphologies of young (1-3 GEN), 5 GEN, and old (10 GEN) cells were visualized by fluorescence microscopy following Mitotracker Green FM staining.(B) Mitochondrial morphologies of young (Y) and old (O) cells of single-deletion strains for DRP fission genes (ΔFis1, ΔMdv1, and ΔDnm1) and overexpression strain of the DRP fusion gene (Fzo1-OE) were also assessed using the same method.(C) Mitochondrial morphologies of young and old Cn cells grown in the presence of 50 µM MitoTEMPO, a mitochondrially targeted antioxidant, were visualized using the same method.Scale bars in white = 5 µm.

FIG 4 A
FIG 4 A suite of cellular antioxidant defense mechanism regulates oxidative stress in Old Cn cells.(A) Cellular ROS, (B) mitochondrial ROS, and (C) mitochondrial calcium levels of young and old cells were measured using a fluorescence plate reader by quantifying the fluorescent intensity of H 2 DCFDA, MitoSox Red, and Fluo-4 AM, respectively.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (**P < 0.01, ***P < 0.001, and ****P < 0.0001).(D) mRNA expression of superoxide dismutase genes (SOD1 and SOD2) was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in young (gray bars) and old (red bars) cells.Bars signify the mean ± SEM of biological triplicates, and student's t test was performed to determine the P-value (**P < 0.01).The mRNA expression is presented as log 2 FC relative to the expression of ACT1, which was used as an internal control.(E) Cellular localization of SOD2-GFP in young and old cells was visualized by fluorescence microscopy.Cells were stained with Mitotracker Red to check the co-localization of SOD2-GFP with the mitochondria.Scale bars in white = 5 µm.(F) mRNA expression of antioxidant genes was analyzed by RT-qPCR in young (gray bars) and old (red bars) cells.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (**P < 0.01and ***P < 0.001).The mRNA expression is presented as log 2 FC relative to the expression of ACT1, which was used as an internal control.

FIG 5
FIG 5 Old Cn cells display changes to ABC transporters and ergosterol synthesis.(A) mRNA expression of genes encoding ABC transporters was analyzed by RT-qPCR in young (gray bars) and old (red bars) cells.The mRNA expression is presented as log2FC relative to the expression of ACT1, which was used as an internal control.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (*P < 0.05, ***P < 0.001, and ****P < 0.0001).ABC transporter activities of young and old cells were measured by quantifying the efflux of (B) rhodamine 6G and (C) Nile Red using a fluorescence plate reader over 30 min.Markers signify the mean ± SEM of biological triplicates (*P < 0.05 and **P < 0.01).(D) Sterol contents of young and old cells were analyzed by gas chromatography-mass spectrometry.Representative total ion current chromatogram of total sterols, with cholesterol (Chol) and ergosterol (Erg) peaks labeled.(E) Ergosterol levels were quantified and normalized to inorganic phosphate (Pi).Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (*P < 0.05).(F) Sterol levels of young and old cells were measured using a fluorescence plate reader by quantifying the fluorescent intensity of filipin III.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (****P < 0.0001).(G) mRNA expression of ergosterol synthesis genes was analyzed by RT-qPCR in young and old cells, and mean log2FC (Continued on next page)

FIG 5 ( 11 FIG 6
FIG5 (Continued)    comparing expression in old/young cells (with error bars reflecting ±SEM) is shown for each gene.Genes in red indicate upregulation in old with log2FC > 1, while genes in gray indicate downregulation in old with log2FC < −1.Bars signify the mean ± SEM of biological triplicates.Student's t test was performed to determine the P-value for the differences in mRNA expression in young and old cells for each gene, as marked above the graph.

FIG 6 (FIG 7
FIG6 (Continued) 0.05, *P < 0.05, ***P < 0.001, and ****P < 0.0001).(C) mRNA expression of genes encoding ABC transporters was analyzed by RT-qPCR in young (gray bars) and old (red bars) cells grown in the presence of 50 µM MitoTEMPO.The mRNA expression is presented as log2FC relative to the expression of ACT1, which was used as an internal control.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (ns P > 0.05, *P < 0.05, and **P < 0.01).ABC transporter activities of young and old cells grown in the presence of 50 µM MitoTEMPO were measured by quantifying the efflux of (D) rhodamine 6G and (E) Nile Red using a fluorescence plate reader over 30 min.Markers signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (*P < 0.05 and ***P < 0.001).(F) Sterol contents of young and old cells grown in the presence of 50 µM MitoTEMPO were analyzed by GC-MS.Representative total ion current chromatogram of total sterols, with cholesterol (Chol) and ergosterol (Erg) peaks labeled.(G) Ergosterol levels were quantified and normalized to inorganic phosphate (Pi).Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value.(ns P > 0.05).(H) Sterol levels of young and old cells grown in the presence of 50 µM MitoTEMPO were measured using a fluorescence plate reader by quantifying the fluorescent intensity of filipin III.Bars signify the mean ± SEM of biological triplicates, and Student's t test was performed to determine the P-value (ns P > 0.05).(I) mRNA expression of ergosterol synthesis genes was analyzed by RT-qPCR in young and old cells, and mean log2FC comparing expression in old/young cells grown in the presence of 50 µM MitoTEMPO (with error bars reflecting ±SEM) is shown for each gene.Genes in red indicate upregulation in old with log2FC > 1, while genes in gray indicate downregulation in old with log2FC < −1.Bars signify the mean ± SEM of biological triplicates.Student's t test was performed to determine the P-value for the differences in mRNA expression in young and old cells for each gene, as marked above the graph.

TABLE 1
Subset of genes upregulated in old