Accumulation of Trehalose 6-Phosphate in Candidozyma auris results in Decreased Echinocandin Resistance and Tolerance

Domain: biology
Published: 2025-12-13
Editorial outcome: minor_revision

Abstract
Candidozyma auris is an emerging multidrug-resistant fungal pathogen that poses a major public-health challenge owing to high mortality and the limited efficacy of current therapies. Echinocandins, which inhibit β-glucan synthesis, are first-line therapy for invasive C. auris infection; however, resistance to this class is rising, underscoring the urgent need for new antifungal targets. Here we show that enzymes in the trehalose-biosynthetic pathway regulate stress responses, antifungal resistance/tolerance and virulence in C. auris. The tps2Δ strain displays heightened susceptibility to echinocandins, whereas tps1Δ and tps1Δ tps2Δ strains show resistance and tolerance comparable to wild type (WT). Mechanistically, the tps2Δ strain accumulates trehalose 6-phosphate (T6P), which inhibits hexokinase activity and reduces the flux of glucose 6-phosphate (G6P) into the chitin biosynthesis pathway, leading to substantially decreased cell wall chitin. During echinocandin exposure, the tps2Δ strain fails to compensate for reduced β-glucan with increased chitin, thereby rendering it highly susceptible to these drugs. In a systemic mouse infection model, deletion of the TPS2 gene results in lower tissue fungal burdens after treatment with caspofungin. Together, these findings identify Tps2 as a potential therapeutic target that can potentiate echinocandin efficacy in C. auris via a distinct mechanism of action.

Introduction
Candidozyma auris is an emerging opportunistic pathogen known for its ability to colonize the skin and cause life-threatening systemic infections, particularly in hospitalized patients with underlying health conditions, including those affected by COVID-191,2. What makes C. auris particularly concerning is its frequent resistance to multiple classes of antifungal drugs, including azoles, polyenes, and echinocandins, which significantly complicates treatment3,4. Studies have shown that up to 90% of C. auris isolates exhibit resistance to fluconazole, 50% to amphotericin B, and 5% to echinocandins5,6. In vivo studies indicate that micafungin, an echinocandin, is more effective in treating invasive C. auris infections compared to fluconazole or amphotericin B7. However, the widespread use of these echinocandins has raised concerns about the potential development of resistance and tolerance to this critical class of antifungal drugs8–11. Here, resistance is considered as a decrease in susceptibility—i.e., an increased MIC relative to a control or clinical breakpoint—caused by stable genetic changes that alter the drug target and/or intracellular drug concentration. In contrast, tolerance is the ability of a otherwise drug-susceptible isolate to survive and grow slowly at concentrations above the MIC without a change in MIC. Tolerance reflects phenotypic heterogeneity and is typically mediated by central stress-response pathways12.

During infection, C. auris faces numerous stressors, including temperature fluctuations, nutrient and osmolarity changes, and hypoxic or oxidative stress, all of which can impact its survival and virulence13–15. The yeast must efficiently mobilize its energy reserves and protect its proteins against these damaging insults. Notably, the disaccharide trehalose has been implicated in providing protection against these stresses, playing a crucial role in the survival mechanisms of yeast under hostile conditions. Understanding the molecular basis of these protective strategies in C. auris is essential for developing new therapeutic approaches to combat this formidable pathogen16–18.

Trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules linked by an α,α-1,1-glycosidic bond and is synthesized by a wide range of organisms, including bacteria, fungi, plants, and invertebrates17,19. However, trehalose is notably absent in mammals. In pathogenic fungi, trehalose plays a crucial role in cellular protection and stabilizing membranes under various stress conditions encountered during infection, including oxidative stress, osmotic stress and heat shock11. In addition, the protective effects of trehalose are also attributed to its ability to stabilize proteins by preventing their denaturation and aggregation20–23. Moreover, trehalose aids in the reactivation of denatured proteins by facilitating their interaction with molecular chaperones, such as heat shock proteins24. In addition to its protective functions, trehalose has been proposed to act as a reserve carbon source25. The biosynthesis of trehalose is governed by a two-step enzymatic pathway26. The first enzyme, Tps1, catalyzes the conversion of uridine diphosphate glucose (UDPG) and glucose 6-phosphate (G6P) into trehalose 6-phosphate (T6P). This T6P has been implicated in the regulation of hexokinase II, which in turn influences glycolytic flux in fungi27–29. In the next step, the second enzyme, Tps2, removes the phosphate group from T6P to produce trehalose. This biosynthetic pathway is absent in humans, making it an attractive target for the development of novel antifungal therapies25,30. Targeting the trehalose biosynthesis pathway in fungi could disrupt key stress responses and virulence mechanisms25, thereby providing a means for combating fungal infections with potentially minimal off-target effects in humans.

In this study, we investigated the role of the trehalose biosynthesis pathway in drug resistance and pathogenicity of C. auris. Our findings highlight the importance of trehalose biosynthetic enzymes in supporting fungal growth under thermal stress and in modulating antifungal drug responses. We found that the accumulation of T6P plays a critical role in inhibiting hexokinase activity, thereby reducing glycolytic flux and impairing cell wall biosynthesis. This disruption compromises the cell’s ability to compensate for echinocandin-induced cell wall damage, leading to decreased antifungal resistance and tolerance. These findings highlight Tps2 as a promising target for antifungal drug development.

Results
Tps2 is important for heat and cell wall stress in C. auris
To assess the role of the trehalose biosynthesis pathway during growth of C. auris, we generated single-gene deletion mutants of TPS1 (tps1Δ) and TPS2 (tps2Δ), as well as a double mutant (tps1Δ tps2Δ), in the clade I reference strain B8441. Growth curve assays were performed in RPMI 1640 medium supplemented with either 0.2% or 2% glucose. Unlike what has been observed in other yeast species31–33, disruption of the trehalose biosynthesis pathway in a C. auris clade I strain did not impact growth under physiological glucose concentrations, as all mutant strains (tps1Δ, tps2Δ, and tps1Δ tps2Δ) exhibited growth profiles comparable to the WT strain under both glucose concentrations (Fig. 1A). These results highlight a distinct metabolic feature of C. auris and indicate that trehalose biosynthesis is not essential for its glucose metabolism or overall growth under these tested conditions.

Given the established role of trehalose in mediating environmental stress responses, we evaluated the growth and stress susceptibility of the trehalose biosynthesis-deficient strains under various conditions. To assess thermal stress tolerance, growth assays were conducted at 37, 39, and 42°C. All mutant strains exhibited varying degrees of growth inhibition, with the tps2Δ strain exhibiting the most severe defect. Specifically, tps2Δ displayed impaired growth at 39°C and completely failed to grow at 42°C, indicating a marked loss of high-temperature tolerance (Fig. 1B). In contrast, although the tps1Δ and tps1Δ tps2Δ strains also exhibited reduced growth relative to the WT at 42°C, their growth was less affected than that of the tps2Δ strain. These results suggest that trehalose synthesis plays a critical role in maintaining thermal stress tolerance in C. auris.

To evaluate stress resistance, the mutant and WT strains were tested against oxidative stress (hydrogen peroxide, H₂O₂), osmotic stress (sodium chloride, NaCl), membrane stress (sodium dodecyl sulfate, SDS), and cell wall stress (calcofluor white, CFW, and Congo red, CR). No significant differences were detected between the WT and mutant strains under oxidative, osmotic, or membrane stress conditions (Fig. 1C). In contrast, clear differences emerged under cell wall stress. The tps2Δ strain showed increased sensitivity to CFW, a chitin-binding dye, but exhibited enhanced resistance to CR, which binds to β-glucan in the cell wall. By comparison, tps1Δ and tps1Δ tps2Δ strains did not display significant differences in sensitivity to either CFW or CR relative to the WT strain (Fig. 1C).

To quantitatively assess stress resistance, we calculated the area under the growth curve (AUC) for each condition and normalized these values to the WT strain to obtain relative resistance values (RRAUC), with the WT defined as 1. Values above 1 indicate greater resistance than WT, while values below 1 indicate reduced resistance. The relative ratios (RR) of AUC were calculated in Eq. 1:

RRAUC = AUC(mutant strain) / AUC(WT) (1)

These values are summarized in a heatmap (Fig. 1D). The tps2Δ strain exhibited reduced resistance to CFW and H₂O₂, but increased resistance to CR compared with the WT strain. In contrast, both tps1Δ and tps1Δ tps2Δ strains showed decreased resistance to both CFW and CR relative to the WT strain.

To confirm that the observed phenotypes were attributable to loss of the TPS2 gene, we complemented the tps2Δ strain to generate a TPS2-reintegrant strain (tps2Δ+TPS2). Complementation restored the stress-associated growth phenotypes: thermotolerance returned to WT levels, and resistance to the cell wall stressors CFW and CR was largely indistinguishable from the WT strain (Supplementary Fig. S1).

Tps2 plays a role in echinocandin resistance and tolerance
Given the trehalose biosynthetic pathway is involved in cell wall stress response, we investigated its contribution to resistance against clinically relevant antifungal drugs, which target either the fungal cell membrane (azoles and polyenes) or the cell wall (echinocandins). We conducted drug broth microdilution assays (BDA) (Fig. 2A, B) and ETEST® assays (Fig. 2C) to determine resistance and tolerance towards these drugs.

In this context, reduced relative resistance is interpreted as increased susceptibility compared with the WT strain, while tolerance refers to the ability of a subpopulation of cells to grow slowly but persistently at drug concentrations above the minimum inhibitory concentrations (MICs). Together, these assays allowed us to evaluate both resistance and tolerance phenotypes in the trehalose-deficient mutants.

Results from the BDAs revealed that deletion of the TPS2 gene significantly reduced resistance and tolerance to all three echinocandins (caspofungin, micafungin, and anidulafungin). This is evident from respectively lower MIC and reduced growth at concentrations above the MIC (supra-MIC growth), quantified by lower AUC of the BDA (Fig. 2A). RRAUC calculated from the growth curves serve as an indicator of drug tolerance, which is visualized in the heatmap (Fig. 2B). The tps2Δ strain displayed a complete loss of tolerance to all three echinocandins and slightly increased tolerance to fluconazole. Meanwhile, the tps1Δ and tps1Δ tps2Δ strains showed a slight increase in tolerance to amphotericin B.

Consistent with the phenotypic observations, Etest analysis demonstrated a clear reduction in echinocandin resistance and tolerance in the tps2Δ strain compared to the WT. The MIC values for all three echinocandins were lower in the tps2Δ strain than in the WT, indicating increased susceptibility. The specific MIC values are shown in the upper right corner of each Etest. Furthermore, the tps2Δ strain exhibited minimal to no growth within the inhibition elipse after 48 h, reflecting a complete loss of echinocandin tolerance (Fig. 2C). In contrast, no significant differences in susceptibility or tolerance to amphotericin B or fluconazole were observed between the tps2Δ and WT strains.

To further establish that the phenotype stems from TPS2 gene loss, we assessed caspofungin susceptibility and tolerance in the TPS2 reintegrant strain (tps2Δ+TPS2) using BDA and Etest assays (Supplementary Fig. S2). Complementation restored WT behavior: the caspofungin MIC of tps2Δ+TPS2 matched WT, and supra-MIC growth was likewise comparable. These results confirm that the heightened echinocandin sensitivity of the tps2Δ strain is due to TPS2 gene deficiency.

T6P accumulation in the tps2Δ strain is independent of caspofungin treatment
To investigate whether intracellular levels of trehalose and T6P influence echinocandin resistance and tolerance in C. auris, we quantified these metabolites in exponentially growing cells, both in the presence and absence of caspofungin (Fig. 3A, B). In unstressed conditions, deletion of the TPS2 gene led to a substantial accumulation of T6P, reaching levels nearly 300-fold higher than those observed in the WT strain. Conversely, deletion of the TPS1 gene, either alone or in combination with the TPS2 gene, resulted in a complete loss of detectable trehalose or T6P. Interestingly, deletion of the TPS2 gene alone still results in low accumulation of a small, residual amount of trehalose, similar to what is observed in C. albicans and C. glabrata31.

To assess whether trehalose or T6P levels respond to echinocandin stress, exponentially growing cells were treated with caspofungin at ten times MIC (2.5 µg/mL) for 60 min. However, no significant changes in trehalose or T6P levels were observed in either the WT or mutant strains following treatment (Fig. 3A, B). It is interesting to note that in the WT under normal conditions, there is already a high amount of trehalose produced. This is different to e.g., the situation in S. cerevisiae, where in exponentially grown cells, there is nearly no accumulation of trehalose34. This difference may be responsible for the intrinsically very high stress tolerance of C. auris.

Tps2 exhibits conserved roles across distinct C. auris clades
To assess whether the role of the trehalose synthesis pathway is conserved across different clades of C. auris, we performed a phylogenetic analysis of the Tps2 proteins (Fig. 4A), which revealed that Tps2 is highly conserved across all five C. auris clades, with minimal sequence divergence. To further investigate the functional conservation of the TPS2 gene, we generated TPS2 gene deletion strains in representative strains from Clade III (B11221), Clade IV (B11244), and Clade V (IFRC2087). Clade II was excluded from this analysis, given that Clade II strains are rarely associated with systemic infections and echinocandin treatment35.

The resulting tps2Δ strains from different C. auris clades were analyzed using drug and stress BDAs to assess their responses to diverse antifungal treatments and environmental stresses. From the drug BDA assay (Fig. 4B), all tps2Δ strains exhibited reduced MIC values for three echinocandins compared to their respective WT strain. The stress-response phenotypes observed in these tps2Δ strains were also largely consistent with those observed for the Clade I strain B8441. Specifically, deletion of the TPS2 gene resulted in increased sensitivity to CFW and enhanced resistance to CR, indicating alterations in cell wall stress responses.

To further quantify differences in stress and drug tolerance, relative resistance was assessed by calculating the area under the curve (RRAUC) for each condition (Fig. 4C). Across all clades, tps2Δ strains showed a marked reduction in RRAUC values for echinocandins. For CR stress, RRAUC values increased in all tps2Δ strains, while under CFW stress, RRAUC values decreased, further supporting a shift in cell wall composition and stress response. These findings demonstrate that the function of Tps2 in supporting echinocandin resistance and tolerance and maintaining cell wall integrity is conserved across multiple C. auris clades.

Deletion of the TPS2 gene results in impaired chitin biosynthesis
To investigate the mechanism underlying the increased echinocandin susceptibility of the tps2Δ strain, we compared cell-wall composition in the WT Clade I isolate B8441 and the corresponding panel of Clade I mutant strains. Confocal microscopy was performed to visualize specific components of the cell wall, employing CFW to detect chitin, Alexa Fluor 488–conjugated antibodies to stain β-glucans, and Concanavalin A–Alexa Fluor 488 to visualize mannans (Fig. 5A). The confocal microscopy images revealed significantly reduced CFW staining intensity in the tps2Δ strain. In contrast, no significant differences in outer cell membrane mannan and β-glucan levels were detected between the tps2Δ and WT strains. The cell wall stainings of tps1Δ and tps1Δ tps2Δ strains showed no notable differences compared to WT cells, indicating no significant alterations in chitin, β-glucan, or mannan content.

To further substantiate the microscopy findings, we quantified the same three major cell wall components using high-performance liquid chromatography (HPLC). These data confirmed a significant reduction in chitin content in the tps2Δ strain compared to the WT (Fig. 5B). In contrast, mannan and β-glucan levels remained comparable across all strains.

The observed reduction in chitin content in the tps2Δ strain prompted us to investigate basal expression levels of genes involved in fungal cell wall biosynthesis (Fig. 6A). To this end, we examined the expression of several putative chitin synthase genes (CHS1, CHS2, CHS3, and CHS8) (Fig. 6B), as well as the GFA1 gene (Fig. 6C), which encodes glutamine-fructose-6-phosphate amidotransferase—an enzyme catalyzing the initial step in the hexosamine biosynthetic pathway essential for chitin synthesis. In addition, we assessed the expression of genes encoding β-glucan synthases (FKS1 and FKS2) to comprehensively evaluate cell wall biosynthesis alterations in mutant strains (Fig. 6D).

Quantitative PCR (qRT-PCR) analysis revealed significant alterations, primarily within the chitin biosynthesis pathway. Notably, GFA1 expression was significantly increased in all mutant strains relative to the WT, underscoring a critical link between trehalose metabolism and chitin biosynthesis. Conversely, CHS1 and CHS2 genes expression levels were significantly decreased in the tps2Δ strain, while expression levels of CHS3 and CHS8 genes remained unchanged across all strains. Furthermore, expression of FKS1 and FKS2 genes showed no significant differences, suggesting that β-glucan synthesis was unaffected by TPS1 or TPS2 gene disruption. Collectively, these results are consistent with and support the observed reduction in chitin content specifically observed in the tps2Δ strain.

Gfa1 is essential but does not account for the tps2Δ echinocandin susceptibility
Gene expression assays revealed that GFA1 gene expression was upregulated in the tps2Δ strain. Based on this observation, we initially hypothesized that T6P accumulation in the tps2Δ background may directly alter the structure or enzymatic activity of Gfa1, thereby impairing chitin synthesis and compromising cell wall integrity, ultimately rendering the cells more susceptible to echinocandin-induced stress. To test this hypothesis, we generated a GFA1 gene knockout strain in the B8441 background and assessed its phenotype using BDA and Etest analysis. Growth curve analysis demonstrated that Gfa1 is essential for viability with glucose as a carbon source in C. auris, as deletion mutants were unable to grow in glucose-containing media (Fig. 7A). The restoration of growth upon supplementation with exogenous glucosamine confirmed that the growth defect was attributable to impaired hexosamine biosynthesis. Notably, when glucosamine was provided at a minimal concentration (10 mM) sufficient to support basal growth, the gfa1Δ strain exhibited substantially reduced resistance and tolerance to caspofungin (Fig. 7B, C). Increasing the concentration of glucosamine progressively improved both resistance and tolerance; however, full recovery to WT levels was not achieved. These findings indicate that the accumulation of T6P does not directly alter the structure or enzymatic function of Gfa1.

T6P accumulation indirectly reduces chitin production by inhibiting hexokinase activity
To investigate how the deletion of the TPS2 gene affects the chitin synthesis pathway, we focused on the role of T6P, a known regulator of the initial steps of glycolysis in yeast, primarily through its inhibition of hexokinase II (Fig. 8A)36. To assess whether this is also the case for C. auris, we analyzed hexokinase enzymatic activity in WT and mutant strains. Both the tps1Δ and tps1Δ tps2Δ strains exhibited increased hexokinase activity, while deletion of the TPS2 gene resulted in a significant reduction in hexokinase activity compared to the WT strain (Fig. 8B). Complementation of TPS2 gene restored hexokinase activity to WT levels (Supplementary Fig. S3).

Considering that the trehalose biosynthesis pathway modulates glycolytic flux in various fungal species, we subsequently measured metabolites derived from glucose-dependent pathways. Therefore, 13C NMR spectroscopy was conducted to elucidate the interplay between glycolysis, trehalose metabolism, and chitin synthesis (Fig. 8C–F). The analysis revealed that the tps1Δ and tps1Δ tps2Δ strains exhibited an increased and quicker glucose consumption compared to the WT (Fig. 8C). This is similar to what is observed in S. cerevisiae, where a tps1Δ strain has a very high flux into glycolysis because of very high hexokinase activity. As a result of this higher influx in glycolysis, both the tps1Δ and tps1Δ tps2Δ strains produced ethanol at a higher rate compared to the WT (Fig. 8D), while glycerol and mannitol levels remained unchanged (Fig. 8E, F). In contrast, the tps2Δ strain exhibited a pronounced reduction in glucose consumption (Fig. 7C), accompanied by significantly lower levels of glycolysis-derived metabolites, including ethanol, glycerol, and mannitol. As a result, reduced carbon flow through glycolysis may limit the availability of F6P for downstream biosynthetic pathways. Given that UDP-N-acetylglucosamine (UDP-GlcNAc), the precursor for chitin synthesis, is derived from F6P, these findings also imply that chitin biosynthesis is compromised in the tps2Δ strain. This metabolic constraint likely explains the reduced chitin levels detected in the tps2Δ strain. 13C NMR spectroscopy also confirmed the synthesis of trehalose for the WT and accumulation of T6P for the tps2Δ strain (Supplementary Fig. S4).

Exogenous glucosamine restores caspofungin resistance and tolerance in the tps2Δ strain
To determine whether the reduced resistance and tolerance of the tps2Δ strain to echinocandins were directly attributable to T6P accumulation, resulting in impaired glycolytic flux and subsequently diminished chitin biosynthesis, we performed caspofungin susceptibility assays with varying concentrations of exogenous glucosamine to evaluate its potential to rescue the susceptibility phenotype. The results demonstrated a non-linear relationship between glucosamine concentration and caspofungin resistance. Specifically, the MIC values of the tps2Δ strain were approximately 0.125 µg/mL at 10 mM, 0.25 µg/mL at 20 mM, and 1 µg/mL at both 30 mM and 50 mM glucosamine. These data suggest that low to moderate levels of glucosamine partially restore caspofungin tolerance in the absence of the TPS2 gene (Fig. 9A).

Consistent with the BDA findings, Etest assays performed after 48 h confirmed that glucosamine supplementation improved caspofungin resistance and tolerance in the tps2Δ strain, but this improvement did not continue beyond 30 mM. At both 30 mM and 50 mM glucosamine, the tps2Δ strain exhibited similar capacity for growth within the inhibition ellipse (Fig. 9B). Together, these results suggest that while glucosamine can compensate to some extent for the loss of the TPS2 gene, its ability to enhance caspofungin resistance and tolerance is limited and does not increase indefinitely with concentration.

Deletion of Tps2 sensitizes C. auris to caspofungin in a murine candidemia model
Given the pronounced defects in echinocandin resistance and tolerance observed in the tps2Δ strain under in vitro conditions, we determined whether these phenotypes translated to differences in susceptibility in an in vivo context. To assess the impact of the tps2Δ strain on the in vivo susceptibility of C. auris to caspofungin, immunocompromised mice were intravenously infected with either the WT strain or the tps2Δ strain and subsequently treated with caspofungin or PBS. To assess the virulence of C. auris strains and the therapeutic impact of caspofungin, we measured fungal burden in the kidneys and brains of infected mice, as schematically depicted in Fig. 10A. To assess the health status of mice after infection and treatment, body weight was monitored daily from day 0 to day 6 (Fig. 10B). No significant differences in body weight were observed among the groups, regardless of infection with WT or tps2Δ strains, or treatment with PBS or caspofungin. Further, we found that in the absence of caspofungin treatment, both the WT and tps2Δ strains showed similar levels of fungal burden in these organs, indicating that deletion of the TPS2 gene does not impair the intrinsic virulence of C. auris. However, following caspofungin treatment, fungal burden in mice infected with the tps2Δ cells was significantly lower than in those infected with the WT strain in both kidneys and brains (Fig. 10C). This indicates that tps2Δ cells are less able to survive after caspofungin treatment in vivo. Together, these findings suggest that loss of the TPS2 gene leads to a complete loss of caspofungin tolerance under host conditions and that a combination therapy of echinocandin with a Tps2 inhibitor would be an effective combination therapy.

Methods
Strainsandgrowthconditions
TheparentalstrainsusedinthisstudyweresinglecolonyisolatesfromclinicalstrainsofPakistan(CladeI)8,SouthAfrica(CladeIII)8,Colombia(CladeIV)72 and Iran (Clade V)73. The strain ID information of each parentalstrainislistedinSupplementaryData1.Strainswerestoredat−80°Cin20%glycerolandroutinelyculturedonsolidYPDagar(1%yeastextract,2%bacteriologicalpeptone,2%dextroseand2%agar)at37°C.Unlessotherwisestated,C.auriscellswereculturedin5mLofRPMI 1640 with L-glutamine (Sigma-Aldrich, Saint Louis, MO, USA) bufferedwith0.165Mmorpholinepropanesulfonicacid(MOPS)topH7andsupplementedwith2%glucose.Cultureswereincubatedat37°Cin a shaking incubator set at 250rpm. During the transformations,nourseothricin(NAT)(WERNERBIO)wasaddedtothemediumtoafinalconcentrationof200µg/mLtoselectforcellswhichcontainedtheNATmarker.

Plasmidconstruction
Plasmids were propagated in E. coli TOP10F′ chemically competent cells.Bacterialtransformationwasperformedbyheatshockat42°Cfor45susing30µLofcompetentcells,followedbyincubationonicefor2min.TransformantswereselectedonLBagar(LBpowder,SigmaorFisherScientific;1.5%[w/v]Bacto™Agar,BD)supplementedwithampicillin (100µg/mL). To generate the deletion mutants, we employed the SAT1 flipper system74. Specifically, the upstream and downstreamregionsofthetargetgenes(TPS1,TPS2andGFA1)wereamplifiedfromthegenomicDNAofC.aurisstrainB8441.These regions were directionally cloned into the pSFS2 vector ina homodirectional way74, flanking the NAT marker and the gene encoding recombinase. The upstream regions were inserted into ApaI/XhoI digested pSFS2 using NEBuilder HiFi DNA Assembly, following the manufacturer’sprotocol.Theresultingconstructswereisolatedfromthe transformants, digested with SacII and NotI-HF, and the downstreamregionsweresubsequentlyclonedintothepreparedplasmids.All inserts of the plasmids were verified by sequencing (Mix2Seq,EurofinsGenomics)toconfirmcorrectintegration.AllplasmidsusedinthispaperlistedinusingtheprimerslistedinSupplementaryData1.Finally, linear deletion cassettes were produced by digesting each plasmidwithKpnI-HF/BglI(NEB).

Strainconstructionandgenedeletion
Trehalose metabolism deletion mutants were generated in C. auris strains B8441(Clade I, WT), B1122 (Clade III), B11244 (Clade IV) and IFRC2087(CladeV)backgroundstrains.TheywereinitiallyculturedovernightinYPDmediumat37°Cwithshaking.Theovernightculturewas then diluted to OD 0.4 and grown until reaching an OD 1.5–2.0.Cellswereharvestedbycentrifugation(3500xg,5mins)andresuspended in transformation buffer (1 x TE buffer, and 100mM lithium acetate (LiAc)), followed by incubation at 37°C for 1h with shaking.Subsequently,1Mdithiothreitol(DTT)wasadded,andcellswereincubatedforanadditional30min.Thecellswerethenwashedwithice-colddistilledwaterand1Msorbitolandfinallyresuspendedin1M sorbitol to prepare competent cells. Transformation was performedviaelectroporationusingatransformationmixturecontainingAlt-R™S.p.Cas9NucleaseV3,duplexedgene-specificAlt-R®CRISPR-Cas9 crRNA (IDT) with Alt-R® CRISPR-Cas9 tracrRNA (IDT), gene-specificguideRNA(gRNA)(IDT),andthelinearizedconstructedpSFS2 variantforeachgeneasdonorDNA.Followingelectroporation,cellswereincubatedinYPG(3% glycerol) recovery medium and subsequentlyplatedontoYPGagarsupplementedwithNATforselection.SuccessfulgenedeletionswereverifiedbycolonyPCRusingprimersflankingtheupstreamanddownstreamtargetgeneregions.Detailsofthe primer sequences and crRNA design are provided in the primers listed in Supplementary Data 2. For double gene deletion, verifiedsingle-genedeletiontransformantswereculturedovernightinYPmediumcontainingmaltoseat37°CtoinduceexcisionoftheNATresistancecassette.Thesametransformationprotocolwasthenusedto generate the second gene deletion, following identical steps asdescribedabove.ForGFA1genedeletion,followingelectroporation,cellswererecoveredinYPDmediumsupplementedwith20mMglucosamine.Followingrecovery,cellswereplatedontoYPDagarcontaining20mMglucosamineandNATforselectionoftransformants.

For reintegration of the TPS2 gene into the B8441 tps2Δ background,a singletps2Δisolate wasused. Deletion mutantswereculturedinYPDandrepeatedlystreakedtoobtainclonesthathadlosttheNAT resistance cassette; cassette excision was verified by PCR. The residualFRTscarwasthentargetedusingtheepisomalCRISPR–Cas9 vectorpJMR19(fromRybaklab)75,enablingscarlessrestorationofthenative TPS2 locus without introducing permanent exogenous sequences.AdonorfragmentcontainingtheTPS2genetogetherwith~500bpofupstreamanddownstreamgenomicsequencewasamplifiedfromB8441genomicDNAandusedforhomologousrecombination. Transformants were selected on YPD supplemented with NAT(200µg/mL)andscreenedbydiagnosticPCRusingprimersbindingoutside the homology arms and within the TPS2ORF. Correct integrationwasconfirmedbyPCRacrossboththe5′and3′junctionsandsubsequentlybysequencing.

Growthphenotypeassay
ThegrowthdynamicsofC.aurisstrainsweremonitoredovertimeinliquid RPMI 1640 medium. Overnight cultures of each strain werewashedthreetimeswithsterileMilli-QwaterandsubsequentlydilutedtoaninitialOD of0.1.Then,20µLofthecellsuspensionwasaddedto180µLofRPMI1640mediumsupplementedwitheither0.2%or2%glucoseasthecarbonsource.Growthwasassessedat37°C,39°C,and42°CbymeasuringOD every30minusingaMultiskanFCmicroplatephotometer(ThermoScientific)inflat-bottom96-wellmicrotiterplates (Bioké). During incubation, cultures were subjected to intermittent pulsed shaking (1min duration every 10min at mediumstrength)toensureaerationanduniformcelldistribution.DataanalysiswasperformedinGraphpadPrismversion10.4.2.

Drugandstresssusceptibilitytesting
The susceptibility of C. auris strains to various antifungal agents—caspofungin, micafungin, anidulafungin, amphotericin B, and fluconazole(allsourcedfromMerck)—wasevaluatedbybrothdilutionassayaccording to guidelines provided by the Clinical and LaboratoryStandards Institute (CLSI)76. In brief, antifungal agents were seriallydilutedtwo-foldinRPMI1640mediumbufferedwithMOPS(pH7.0)supplementedwith2%glucoseand1%DMSO,reachingafinalassayvolumeof200µLperwell.Theconcentrationrangetestedwasfrom16to 0.0625µg/mL for caspofungin, micafungin, amphotericin B, and fluconazole, and from 8 to 0.03125µg/mL for anidulafungin. Subsequently, an inoculum containing ~25,000 cells/mL was dispensed into round-bottom 96-well polystyrene microtiter plates(GreinerBio-One)priortoincubation.

StresssusceptibilitywasevaluatedusingthesameBDAprotocol.Stressorconcentrationsinthefinalassaymediumwerepreparedintwo-fold serial dilutions as followed: H2O2 (Sigma-Aldrich) at concentrations ranging from 20 mM to 0.0781 mM, CFW (FluorescentBrightener28,Sigma-Aldrich)atconcentrationsrangingfrom5mMto 0.0195 mM, and CR (Sigma-Aldrich) at concentrations rangingfrom 12mM to 0.0467mM; sodium chloride (NaCl; Sigma-Aldrich)from 2.5M to 0.0098M; and SDS (Sigma-Aldrich) from 3.74mM to 0.014mM.

Plates were incubated at 37°C for 48h. Fungal growth wasquantifiedbymeasuringOD usingaSynergy™H1microplatereaderusingthe(BioTek Instruments). MICs from BDA were determined based on a50%reductioningrowthrelativetothedrug-freecontrol.

In addition to BDA, antifungal susceptibility was also assessedusing ETEST® strips (bioMérieux), following the manufacturer’sinstructions.MICvaluesweredefinedbythepointatwhichtheellipseof inhibition intersected the scale on the strip after 48h of incubationat37°C.

Determinationofintracellulartrehaloselevels
Exponentiallygrowncellswereharvestedbycentrifugation3500xg(4°C)for5min.Thesupernatant was discarded, and the cells were resuspendedin1mLicecoldMilli-Qwater.Next,theywerebrokenbyvigorouslyvortexinthepresenceofglassbeads(0.75-1mm)bymeansof the FastPrep (20s, 6m/s) (MP biomedical™). Then, immediatelytransferredtoa95°Cwaterbathfor5min.Afterwards,thesamplewascentrifugedat7000xg(4°C)for5minand200µLofthesupernatantwasusedforanalysisbytheShimadzuHPLCsystemusinganAgilent87Hcolumnat0.7mL/minandaRID−20Adetector(Shimadzu).1mMsulfuricacidwasusedaseluentataconstantflowof0.6mL/minat80°C.Puretrehalose(Sigma)wasusedasastandard.

DeterminationofintracellularT6Plevels
IntracellularT6PlevelswasmeasuredaccordingtoZhuetal.,202531.Exponentiallygrowncellswereharvestedbycentrifugationat3000xg(4°C)for5min.Weighasquicklyaspossibletogetanaliquotinthe rangeof15-20mg(wetweight).ThecellswereextractedasdescribedinLunnetal.withalittlemodification77.Briefly,cellswererinsedwith175µLice-coldCHCl₃/MeOH(3:7)andincubatedat−20°Cfor2hwithoccasionalvortexing;add350µLice-coldwaterwasadded,vortexed≥5s to disperse phases, warmed to 4°C with intermittent shaking,thencentrifugedat13,000×g,4°Cfor10min.Theupperaqueous/MeOHlayerwastransferredtoanew1.5mLscrew-captubeonice;thelowerCHCl₃phasewasre-extractedwith300µLice-coldwater,vortexedandcentrifugedasabove,andtheupperlayerswerepooledanddriedcompletelyinavacuumcentrifugeat35°C.T6Pwasassayedinextractsbyanion-exchangeHPLCusingaDionexHPLCsystem(Sunnyvale,CA,U.S.A.),coupledtoanABSciexQ-Trap6500triplequadrupolemassspectrometerwithmodificationsasdescribedinFigueroaetal.201678.

RNAExtractionandgeneexpressionanalysis
FortotalRNAisolation,cellsweregrownovernightanddilutedtoanOD of0.2inRPMI1640(2%glucose).Cellsweregrownfor5–7handafterwardswashedice-coldwater,resuspendedin0.5-1 mL TRIzol(ThermoFisher)andbrokenbyusingglassbeadsinaFastPrep machine.RNAwasextractedbytheadditionofchloroformandisopropanol after which three washes with ethanol (70 %) were conducted.RNAwastreatedwithDNaseenzyme(NewEnglandBiolabs)andconvertedtocDNAusingtheiScriptcDNAsynthesiskit(Biorad).qPCR was conducted using GoTaq polymerase (Promega) and theStepOnePlusreal-timePCRdevice(ThermoFisher).Primersusedarelistedinsupplymentarydata2.ACT1,UBC13andRPL10wereusedasreference genes. Data were subsequently analyzed using qBasePlussoftware(Biogazelle)79.

FluorescenceMicroscopytoQuantifyCellWallComponents
Cellswerefixedin50mMThimerosalatroomtemperaturefor24h,washedtwiceinwater,andresuspendedin1mLwater.Forβ-glucanandchitinstaining,approximately2.5×106cellswereincubatedwiththeanti-β-glucanprimaryantibodyFc-Dectin-1(ThermoFisher)(1.5µg/mL)for45minatroomtemperature,followedbyincubationfor30minin the dark with a PBS solution containing GT F(ab’)2 anti-human IgGsecondary antibody conjugated to Alexa Fluor 488 (1:200 dilution)(ThermoFisher)forstainingβ-glucanandCalcofluorWhite(25µg/mL)forstainingchitin.Formannanstaining,2.5×106cellswereseparatelyincubatedfor30mininthedarkwithaPBSstainingsolutioncontainingConcanavalin A-Alexa Fluor 488 (ThermoFisher)(25µg/mL). Afterstaining,cellswerewashedonceinwaterandresuspendedinwaterforanalysis.Formicroscopy,5µLaliquotsweremountedonglassslides,air-dried,andvisualizedusingconfocalmicroscopywithaDAPIfilterset,followingthemethodpreviouslydescribedbyOkadaandOhya80.

Sub-MICCaspofunginTreatmentandChitinStaining
OvernightcultureswerewashedthreetimeswithsterileMilli-Qwater,dilutedtoaninitialOD of0.2,andgrowninRPMI1640supplementedwith2%glucosewiththeindicatedsub-MICconcentrationsofcaspofungin at 37°C for 5-7h with shaking (200rpm). Cells werewashed three times with 1xPBS and fixed in 4% paraformaldehyde(ThermoFisherScientific)at37°Cfor30min.Fixedcellswerewashedthreetimeswith1xPBS,stainedinthedarkwithCFW(10µg/mL)atRTfor30min,washedthreeadditionaltimeswith1xPBS,andimagedbyconfocal microscopy as above. Methods follow our previouslydescribedprotocolofFluorescenceMicroscopytoQuantifyCellWallComponents.

Chemicalquantificationofcellwallcomponents
Cell walls from isolates were prepared from exponentially growingculturesinRPMI1640supplementedwith2%glucose,followingthemethoddescribedbyMunroetal.(2003)81,withminormodifications.Briefly,cellwalls(20mgwetweight)wereextractedbydisruptingcellswith glass beads in a FastPrep machine. Samples were washed fivetimeswith1MNaCl,andcellwallswereextractedusingSDS-Mer-buffer(50mMTris,2%SDS,0.3Mβ-mercaptoethanol,1mMEDTA;pH8.0) at 100°C for10minutes.Cell wall pellets wereresuspended insterileMilli-Qwater,freezedried,andthedryweightofrecoveredcellwallswasmeasured.Chitincontentsweredeterminedbymeasuringthe glucosamine. For glucose and mannose measurement, hydrolysateswerediluted100times(10µLhydrolysatein1mLof2MNaOH)andfurtherdiluted10timeswithMilli-Qwaterinaglassvialforanalysis. Fifteen µL was analyzed by high-performance anion-exchangechromatography with integrated pulsed amperometric detection(HPAEC-iPAD)(ICS−3000,Dionex,Sunnyvale,CA,USA)equippedwithaCarboPacMA1Guardcolumn(50×4)inserieswithaCarboPacMA1column(250×4).Thesugarswereisocraticallyseparatedwith600mMNaOHwithaflowrateof400µL/minof600mMNaOHduring24min.Quantificationwasperformedonpeakareaswithexternalstandardsofglucoseandmannose82,83.

Hexokinaseactivitymeasurement
HexokinaseactivitywasmeasuredaccordingtoWijnantsetal.,2020withsmalladaptations83.Cellsweregrownat37°CinRPMI1640(2%glucose). They were collected and washed twice with 1x imidazolebuffer(50mM,pH7.5,10mMMgCl₂,100mMKCl,and0.1mMEDTA).Thecellpelletwasresuspendedin1ximidazolebuffertoafinalconcentration of 200mg cells/mL. Cells were lysed by FastPrep (20s,6m/s)for10times(placeonicefor30secondsbetweeneachtime)and a protease inhibitor cocktail (complete, EDTA free; Roche) wasadded to prevent proteolysis. The supernatant was collected aftercentrifugationat3000xg (4°C).Hexokinaseactivity was measuredspectophotometrically at340nm every30s for 10min by couplingto the reduction of NADP+ via glucose-6-phosphate dehydrogenase.The reaction contained 2ximidazole buffer, 50mM ATP, 40mM NADP,0.6Uglucose-6-phosphatedehydrogenase(G6PDH;Sigma),2Uphosphoglucoseisomerase(PGI;onlyinthefructosebuffer;Sigma),and0.1%glucoseand20µLofcellextract.TheproteinconcentrationwasmeasuredbytheLowrymethod82.

13CNMRSpectroscopy
Utilizationof13C-labeledglucose([1−13C]glucoseand[u-13C ]glucose,6bothCambridgeIsotopeLaboratories(Andover,MA,USA))wasmonitored by 13C NMR spectroscopy similar to a previously describedstudy84.Inbrief,cellswereculturedinRPMI1640mediumcontaining2%glucoseat37°Cfor5–6hours.Followingincubation,thecellswereharvestedandwashedtwicewith1xPBS.Theywerethenresuspendedataconcentrationof10⁷cells/mLinglucose-freeRPMI1640mediumsupplementedwithMOPSbufferandkeptoniceuntilfurtheruse.ForNMR spectroscopy, cell suspensions (0.4mL) were transferred to5mmNMRtubes(Eurisotop,St.Aubin,France),heatedupto37°Cfor5min and then 0.1mL deuterated water (Eurisotop) containng 13C labeledglucose(finalconcentration100mM)wasadded.NMRtubesweretransferredtotheNMRspectrometerandatimeseriesofNMRspectra was acquired within 5min (n=3-4 per strain). NMR spectra were acquired at 37oC using a Bruker Avance 400MHz NMR spectrometer(BrukerBiospin,Rheinstetten,Germany),equippedwithan5mmbroadbandprobe.Aftertheacquisitionofa1HNMRspectrum(totalacquisitiontime1:30minutes),3or613CNMRspectrawith256averages(totalacquisitiontime15minutes)followedby6or313CNMRspectra with 512 averages (total acquisition time 30minutes) and a final 13C NMR spectrum with 1024 averages (total acquisition time 60min)wereacquired,coveringaperiodofupto4.5h.Otheracquisition parameters were: frequency 100.63MHz, relaxation time 2s,acquisition time 1.5s, spectralwidth 22 000MHz,64 kdata points,protondecouplingusingaWALTZ16sequence.AllNMRspectrawereprocessed and quantified using the Bruker software TopSpin 3.2.Relativequantificationof13Clabeledmetaboliteswasachievedafterphaseandbaselinecorrection,settingthetotalintegralofthealphaandbetaC-1glucosesignalsinthefirst13CNMRspectrumto100%.

Invivoexperiments
Fortheinvivoexperiments,7-week-oldBALB/cfemalemicewereused(Janvier Labs), kept per three in individually ventilated cages. Weacknowledgesexasabiologicalvariableanddiscussthisasalimitation.Foodandwaterwereavailableadlibitum.Forimmunosuppression,cyclophosphamide(CP)wasadministeredviaintraperitoneal(IP)injectionatadoseof200mg/kg3 days prior to infection and150mg/kgthedayaftertheinfection.BothCPinjectionshadatotalvolumeof200μL.Micewereinfectedintravenouslythroughthetailveinwithaninoculumof10⁷C.aurisWTortps2Δcellsin100μLPBS,administeredseparately.TheanimalswereIPtreateddailyfor6days,startingthedayofinfection(2hpostinoculation),with5mg/kgcaspofungin(inatotalvolumeof100μL)orthePBSascontrol.7dayspostinfection, the mice were sacrificed and colonization in kidneysandbrainwasevaluated:organswerehomogenizedin500µLsterile1xPBSwithglassbeadsandshaking(20secondsat6m/second)inaFastPrep−24TM Classic lysis system (MP Biomedicals). The organhomogenateswereseriallydilutedandplatedontoYPDagarforsinglecolony(CFU)isolationafter48hincubationat37°C.

Statistics&reproducibility
The figure legends detail statistical parameters, including the exactsample size (n) and measures of statistical significance. Statisticalanalyses were performed using GraphPad PRISM software version10.4.2(534)(GraphPadetal.,LLC).Thespecificstatisticaltestsappliedarealsodescribedinthefigurelegends.

Ethicalstatement
AllanimalexperimentswereapprovedinaccordancewiththeethicalguidelinesoftheEthicalCommitteeforAnimalExperimentationofKULeuven (project number: 079/2020) and were performed in accordancewiththeregulationsoftheKULeuvenanimalcareguidelines.TheKULeuvenethicalcommitteeforanimalexperimentationispartoftheregionalregulationsimposedbytheFlemishgovernment,Secretary General of the Unit Animal Welfare, ‘Departement Leefmilieu,Natuur&Energie’,KoningAlbertII-laan20bus8,1000Brussel.Allmicewerekeptinfilter-topcagesinananimalroomwithcontrolledtemperature(21°C),humidity(50%~70%),and12hlight/12hdarkcycle.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a really interesting paper where the authors provide convincing in vitro and in vivo evidence that deletion of TSP2 in\nC. auris enhances the susceptibility of C. auris to echinocandins. This indeed suggests that targeting Tsp2 may be a useful\nstrategy to enhance susceptibility of C. auris to echinocandins. I think is definitely something worth pursuing as this multi-\ndrug resistant fungal pathogen continues to spread around the globe.\nMy concerns are these:\n1. When I think about echinocandin tolerance and resistance related specifically to chitin, I attribute tolerance to the natural\nvariations in amount of chitin in the cell walls of cells in a population (i.e. those cells with a naturally high chitin content will\nbe tolerant), but resistance arises due to a stress-induced compensatory increase in cell wall chitin that only occurs when\ncells are treated with a non-lethal dose of echinocandin.\nAlthough the authors show that deletion of TSP2 results in the accumulation of T6P, inhibition hexokinase, reduced flux\nthrough glycolysis, decreased chitin content, and increased susceptibility to echinocandins, they have not actually provided\ndirect evidence that “the inability to compensate the reduction in β-glucan levels with increased chitin production during\nechinocandin treatment in the tsp2Δ strain rendered this strain highly susceptible to these drugs” (line 39-41). Does\ntreatment of C. auris with a sub-MIC of echinocandin result in a compensatory increase in cell wall chitin? Is this\ncompensatory increase no longer observed in a tsp2 mutant?\n2. A decrease in chitin content typically renders cells less susceptible to CFW, not more susceptible. I am therefore finding it\ndifficult to reconcile the phenotype of the tsp2Δ mutant in relation the chitin content and susceptibility to CFW (line 224-226).\nCould the increased susceptibility of the tsp2 mutant to CFW (compared to wildtype) be related more to general stress\nprotection provided by trehalose, rather than chitin?\n3. To my knowledge, the function of the C. auris chitin synthases and their regulation has not yet been investigated.\nHowever, in S. cerevisiae and C. albicans, the class IV chitin synthase (Chs3) makes the majority of cell wall chitin. I would\ntherefore be very cautious of making a statement that decreased CHS1 and CHS2 transcripts in the tsp2 mutant\n(compared to wild type) indicate impaired chitin biosynthesis capacity (line 239).\nMinor comments:\n4. The section “Loss of Tsp2 impairs echinocandin resistance and tolerance via T6P accumulation” (line 163) simply shows\nthat T6P accumulates in the tsp2Δ mutant. The link with echinocandin susceptibility has not yet been established. Perhaps\nthe subheading could be re-worded?\n5. Line 195 and 197: The reference to Figure 1A is incorrect.\n6. Growth phenotype assay (line 546). What volume of culture was added to each well in the 96 well plate? What software\nwas used for the analysis of the growth curves?\n7. The colour and symbol legends in Figures 1 and 2 indicate three tsp1 mutants (-1, -2 and -3), three tsp2 mutants and three\ntsp1tsp2 mutants. Do these refer to biological replicates? Ideally, OD600 should be plotted on a log scale so that the curves\nbetter represent the phases of growth.\n8. Figure 5B: Are the labels on the y-axes correct?", "reviewer_2": "A very interesting paper that investigates the role of trehalose synthesis pathway in Candida auris drug susceptibility, cell\nwall composition stress resistance and virulence. Genetic disruption of the pathway was achieved by deleting the TPS1\nand/or TPS2 genes. The main experiments focussed on mutants generated in Clade 1 strain but to show conservation of the\nphenotype mutants were made in Clade 2, 3, 4 and 5. One limitation of the study is that control reintegrant strains have not\nbeen constructed to show complementation with the WT genes restored WT phenotypes. Nevertheless the study follows a\nlogical line of enquiry and makes steps to understand the underpinning mechanisms contributing to the observed\nphenotypes. The study is highly relevant and novel and is of importance given the widespread drug resistance of this\nrecently emerged pathogen. It also directly links drug tolerance with cell wall composition and metabolic flux and clearly\ndemonstrates the importance of TPS2 in antifungal susceptibility.\nLine 98: you should first mention that you constructed the mutants before describing the growth assays.\nFigure 1: Growth assays are usually reported with y-axis in log scale\nLine 110: mention the temperatures tested in the main text and the temperatures where tps2 mutant had defective growth.\nLine 113: why was the double mutant less affected than the tps2 mutant?\nLine 117: first describe the stresses that were tested and then the results\nLine 132/132 indicate that reduced relative resistance equates to increased susceptibility and add……in comparison to WT.\nLine 137: is involved\nLine 147 & 161: there are some subtle changes in susceptibility of the tps1 and tps1/tps2 double mutant. Better to state that\nrather than no notable changes\nPresume from Figure 5 onwards the analysis was done on the Clade I WT and mutant? Would be worth stating that at the\nstart of these experiments.\nLine 255: interesting finding as the general rule is that elevated chitin leads to CFW sensitivity and low chitin (e.g. in chs3\nmutant) results in CFW resistance. This finding of low chitin and high sensitivity will probably reflect other alterations in the\ncell wall that impact on CFW sensitivity.\nLine 253- Perhaps not surprising that you didn’t see changes in CHS3 expression as it is mainly regulated post-\ntranslationally. It would have been informative to look at changes in gene expression in presence of echinocandin stress.\nAny thoughts on mechanism of down regulating CHS1 and CHS2 expression? Related to elevated T6P levels and turgor\npressure?\nGFA1 gene product is under allosteric regulation through a negative feedback loop regulated by UDP-GlcNAc levels and\nelevated GFA1 levels may partially circumvent the negative feedback leading to higher UDP-GlcNAc providing more\nsubstrate for chitin synthesis. This could be considered in the discussion section.\nFigure 7A the fructose-6-P to hexosamines is a reversible reaction.\nLine 262: glycolysis spelling\nVery nice approach to add exogenous GlcN to bypass the requirement for F6P and show the effect on drug tolerance,\ndid\nyou test whether this restored chitin levels of the tps2 mutant completely back to WT.\nIn vivo testing showed no differences in burdens when WT was compared with mutant (presuming Clade 1 background) but\nwhen mice were treated with caspofungin tps2 mutant had significantly reduced burdens. Was there any difference in\nsurvival? Did the untreated mice succumb to disease and the caspofungin treated survive? At what time point were the\nfungal burdens assessed.\nLine 309: you haven’t directly shown that “tolerance” is lost in vivo only that burdens were reduced. To show loss of\ntolerance the cells from infected organs could have been plated on eTest strip plates.\nLine 323: you haven’t shown directly that UDP-GlcNAc levels are reduced, you have shown that chitin levels are reduced\nand you should also consider that GFA1 expression is elevated. Incidentally increased expression of GFA1 in response to\ncell wall stress has been previously published in S. cerevisiae (PMID: 11895440) and this could be added to the rationale\nbehind investigating this gene. The supplementary data on gfa1 mutant may not be best introduced in the Discussion\nsection, where it comes as a bit of a surprise, could it be included earlier in the Results?\nFigure 10 spelling chitin synthesis, N of N-acetylglucosamine should be in italics.\nLine 680: 3 weeks?\nMethods: some suppliers are missing: e.g. nourseothricin, anti-β-glucan primary antibody Fc-Dectin-1, Concanavalin A-\nAlexa Fluor 488, IgG secondary antibody conjugated to Alexa Fluor 488", "reviewer_3": "This tightly reasoned report presents mechanistic insight into the emerging pathogen Candida auris' insensitivity to\nechinocandin antifungals. Candida auris has become an increasing menace over the last decade due to its resilience to\nantifungals and disinfectants, so on that basis the study is highly significant. Moreover, the findings point to an enzyme with\na causal role, and in this way the authors have a druggable target. This element of the study further increases its\nsignificance.\nA second strength of the study is the thoroughness of the analysis. For example, strains from all four major pathogenic C.\nauris clades are tested. Also, metabolic inferences are pursued and validated with metabolomic characterization. Finally, a\npoint that all readers will appreciate is the simplicity of the explanation at which the authors arrive. It is the kind of outcome\nthat even students in my Introductory Microbiology class could understand and appreciate! In fact, if this work is published in\ntime, I may even include it in a lecture this fall.\nThe one minor experimental criticism that I have is that the most revealing mutant, the tps2Δ strain, was not complemented.\nThere was a G418 marker developed by Santana and O'Meara in Nat Commun. 2021 Dec 10;12(1):7197 that might be\nuseful, or the authors could recycle their NAT marker as they do for other purposes. I do not doubt the overall\nphenotypic results as they stand - the authors make the mutant in multiple strains, for example - but for some of the findings,\nsuch as reduced chitin accumulation and reduced hexokinase activity, complementation would ensure that the conclusions reach the\nhighest standard.\nThe one minor manuscript preparation criticism that I have is that the Discussion and Results sections seem to overlap more\nthan necessary. I wondered if the authors might benefit from merging the two into a single \"Results and Discussion\" format.\nThis issue is more of a matter of taste though, which is why I consider it quite minor.\nMy one strong recommendation with regard to manuscript preparation is that the authors should define clearly what\nresistance and tolerance each mean. These are terms with distinctions that can be a little hazy among a broad audience. I\nam 99% certain that the authors' assays are completely in line with the simple definitions that I always refer to\n(Berman and Krysan, Nat Rev Microbiol. 2020 Jun;18(6):319-331), so I do not question the authors' assays and conclusions;\nmy recommendation is really to make sure the work can be appreciated by a broad readership."}, "author_response": "We thank the reviewer for this insightful comment. As for the first question (Does treatment\nof C. auris with a sub-MIC of echinocandin result in a compensatory increase in cell wall\nchitin? ). Yes — C. auris, similar to other Candida species, is capable of mounting a\ncompensatory increase in cell wall chitin in response to echinocandin treatment at sub-\ninhibitory concentrations. This adaptive response is a well-established mechanism of\nechinocandin tolerance, as it helps maintain cell wall integrity when β-1,3-glucan synthesis is\nimpaired. Notably, Lara-Aguilar et al. (2021) demonstrated that C. auris exhibits a significant\nincrease in chitin content following exposure to high concentrations of caspofungin.\nAlthough this response is less pronounced compared to C. albicans, it supports the presence\nof a conserved, stress-induced chitin remodeling mechanism in C. auris that contributes to\nsurvival under echinocandin pressure.\nTo directly address the reviewer’s questions, we measured chitin levels after sub-MIC\ncaspofungin treatment in WT and tps2Δ strains. Exponentially growing cells were exposed to\n¼ and ½ MIC caspofungin, stained with Calcofluor White, and analyzed by confocal\nmicroscopy. Sub-MIC caspofungin increased chitin in WT cells, and a comparable increase\nwas observed in the tps2Δ mutant. Thus, the compensatory chitin response to echinocandin\nstress is largely Tps2-independent. At higher (supra-MIC) concentrations, tps2Δ growth was\nmarkedly inhibited, precluding reliable chitin assessment.\nWe have incorporated these new data and discussion into the revised manuscript (new\nFigure S5; revised text, lines 493–495 (in clean manuscript)).\nIn parallel, we have observed that supplementation with exogenous glucosamine — a\nprecursor in the chitin biosynthesis pathway — increases caspofungin resistance and\ntolerance in both wild-type and tps2Δ strains. These results clarify that the\nhypersusceptibility of tps2Δ is unlikely due to a failure to trigger chitin remodeling, but\nrather is consistent with a metabolic limitation (reduced glycolytic flux/precursor supply)\nthat compromises cell-wall robustness under drug stress.\n2. A decrease in chitin content typically renders cells less susceptible to CFW, not more\nsusceptible. I am therefore finding it difficult to reconcile the phenotype of the tsp2Δ\nmutant in relation the chitin content and susceptibility to CFW (line 224-226). Could the\nincreased susceptibility of the tsp2 mutant to CFW (compared to wildtype) be related more\nto general stress protection provided by trehalose, rather than chitin?\nResponse: Thank you for raising this important point. We agree that the phenotype of\ntps2Δ—lower chitin content yet heightened sensitivity to calcofluor white (CFW)—does not\nfollow the usual correlation between total chitin levels and CFW susceptibility. Consistent\nwith previous reports 1-3, our data suggest that CFW sensitivity depends on chitin\naccessibility and overall cell-wall organization, not simply on chitin abundance4. We have\nrevised the text to clarify this distinction.\nWe also considered whether loss of the stress-protective function of trehalose could explain\nthe phenotype. However, both tps1Δ and tps1Δ tps2Δ strains, which are similarly defective\nin trehalose biosynthesis, do not display increased CFW sensitivity compared with the wild\ntype (Figure 1C,D). This argues against trehalose depletion as the main cause.\nInstead, we propose that accumulation of trehalose-6-phosphate (T6P) in tps2Δ disrupts\ncell-wall architecture—for example, by altering β-glucan–chitin cross-linking and increasing\nwall porosity—thereby exposing the remaining chitin to CFW and leading to hypersensitivity\ndespite the overall reduction in chitin content.\n3. To my knowledge, the function of the C. auris chitin synthases and their regulation has\nnot yet been investigated. However, in S. cerevisiae and C. albicans, the class IV chitin\nsynthase (Chs3) makes the majority of cell wall chitin. I would therefore be very cautious of\nmaking a statement that decreased CHS1 and CHS2 transcripts in the tsp2 mutant\n(compared to wild type) indicate impaired chitin biosynthesis capacity (line 239).\nResponse: We thank the reviewer for this clarification. In our dataset, CHS1 and CHS2\ntranscripts were reduced in the tps2Δ mutant. While this pattern may suggest a general\ndown-regulation of the chitin-biosynthetic program, we agree that transcript levels alone do\nnot establish reduced chitin synthesis—especially since CHS3, the principal chitin synthase in\nmany Candida species, was unchanged. A plausible explanation is that Chs3 activity is\nregulated mainly post-translationally. Accordingly, we have removed the over-interpretive\nlanguage from the Results (line 245) and now address potential transcriptional and post-\ntranslational regulation of chitin-synthesis enzymes in the Discussion (lines 442–454).\nMinor comments:\n4. The section “Loss of Tsp2 impairs echinocandin resistance and tolerance via T6P\naccumulation” (line 163) simply shows that T6P accumulates in the tsp2Δ mutant. The link\nwith echinocandin susceptibility has not yet been established. Perhaps the subheading could\nbe re-worded?\nResponse: We agree that the original subheading overstated the link between T6P\naccumulation and echinocandin susceptibility. To better reflect the actual results presented\nin this section, we have revised the subheading to:\n“T6P accumulates in the tps2Δ strain independently of caspofungin treatment”\nThis wording more precisely describes our findings and avoids implying a causal link to\nechinocandin susceptibility, which is addressed in subsequent sections.\n5. Line 195 and 197: The reference to Figure 1A is incorrect.\nResponse: We have corrected the reference by removing the citation to Figure 1A. To clarify\nthe data presentation, we have revised Figures 4B and 4C to include the data for clade I WT\nand clade I tps2Δ strains.\n6. Growth phenotype assay (line 546). What volume of culture was added to each well in\nthe 96 well plate? What software was used for the analysis of the growth curves?\nResponse: We have revised the description in the Methods section to include this\ninformation. Specifically, we now state that each well contained a total volume of 200 µL\n(20 µL cell suspension + 180 µL medium), and that growth curve data were analyzed using\nGraphpad Prism version 10.4.2.\n7. The colour and symbol legends in Figures 1 and 2 indicate three tsp1 mutants (-1, -2 and -\n3), three tsp2 mutants and three tsp1tsp2 mutants. Do these refer to biological replicates?\nIdeally, OD600 should be plotted on a log scale so that the curves better represent the\nphases of growth.\nResponse: In Figures 1 and 2, the “-1, -2, -3” labels represent independent biological\nreplicates (separately isolated tps1Δ, tps2Δ, and tps1Δ tps2Δ transformants), each derived\nfrom distinct colonies and cultured independently. We have clarified this in the figure\nlegends section.\nY-axis scaling. We appreciate the suggestion to use a logarithmic y-axis. A log scale is ideal\nwhen complete growth curves (lag, exponential, and stationary phases) are reliably\ncaptured, as in erlenmeyers-based assays. In our study, OD₆₀₀ measurements were obtained\nwith a microplate reader (multiscan), whose dynamic range under our conditions did not\naccurately capture the stationary phase. Because our analyses focus on the lag and early\nexponential phases, a linear y-axis preserves visual resolution in the low-OD range, whereas\na log scale would compress these values without revealing additional phases. For these\nreasons, we have retained the linear scaling.\nIn the reply to referee 2 we have added figures where we have put the Y axis in a log scale.\n8. Figure 5B: Are the labels on the y-axes correct?\nResponse: We thank the reviewer for raising this point. As described in the Methods, chitin\nlevels were quantified as glucosamine equivalents, while β-glucan and mannan were\nquantified as glucose and mannose equivalents, respectively. So the full y-axes should read\nlike: Chitin: µmol GlcN eq./mg wet weight) β-glucan: µmol Glc eq./ mg wet weight) Mannan:\nµmol Man eq./mg wet weight).\nTo avoid cluttering the y-axis, we have kept the labels short (e.g. Chitin [µmol/mg ww]) and\nadded a clear explanation in the figure legend stating that the values represent\nmonosaccharide equivalents derived from standard curves. This ensures accuracy while\nkeeping the figure easy to read.\nfReviewer #2 (Remarks to the Author):\nA very interesting paper that investigates the role of trehalose synthesis pathway in Candida\nauris drug susceptibility, cell wall composition stress resistance and virulence. Genetic\ndisruption of the pathway was achieved by deleting the TPS1 and/or TPS2 genes. The main\nexperiments focussed on mutants generated in Clade 1 strain but to show conservation of\nthe phenotype mutants were made in Clade 2, 3, 4 and 5. One limitation of the study is that\ncontrol reintegrant strains have not been constructed to show complementation with the\nWT genes restored WT phenotypes. Nevertheless the study follows a logical line of enquiry\nand makes steps to understand the underpinning mechanisms contributing to the observed\nphenotypes. The study is highly relevant and novel and is of importance given the\nwidespread drug resistance of this recently emerged pathogen. It also directly links drug\ntolerance with cell wall composition and metabolic flux and clearly demonstrates the\nimportance of TPS2 in antifungal susceptibility.\nResponse: We appreciate the reviewer’s point regarding the absence of reintegrant controls.\nWe did not include the reintegrant strain as we use three independent mutant\ntransformants and did all the experiments with each time the three biological independent\nstrains. To address the comment by the referee, we now also generated a TPS2-reintegrant\nstrain (tps2Δ+TPS2) and re-evaluated key phenotypes. Complementation restored wild-type\nbehavior across assays: thermotolerance, cell-wall stress responses (CFW and CR), and\ncaspofungin susceptibility and tolerance (BDA/Etest). These data demonstrate that the\nphenotypes observed in tps2Δ are specifically attributable to loss of TPS2. The new results\nand figure references have been added to the manuscript (see Fig. S1–S3).\nLine 98: you should first mention that you constructed the mutants before describing the\ngrowth assays.\nResponse: We have revised the Results to first state the construction of the mutants and\nthen describe the growth assays. The updated text now reads(Line 101-105):\nTo assess the role of the trehalose biosynthesis pathway during growth of C. auris, we\ngenerated single-gene deletion mutants of TPS1 (tps1Δ) and TPS2 (tps2Δ), as well as a\ndouble mutant (tps1Δtps2Δ), in the clade I reference strain B8441. Growth curve assays were\nperformed in RPMI 1640 medium supplemented with either 0.2% or 2% glucose.\nFigure 1: Growth assays are usually reported with y-axis in log scale\nResponse: We appreciate the suggestion to use a logarithmic y-axis. A log scale is ideal when\ncomplete growth curves (lag, exponential, and stationary phases) are reliably captured, as in\nerlenmeyers-based assays. In our study, OD₆₀₀ measurements were obtained with a\nmicroplate reader (multiscan), whose dynamic range under our conditions did not\naccurately capture the stationary phase. Because our analyses focus on the lag and early\nexponential phases, a linear y-axis preserves visual resolution in the low-OD range, whereas\na log scale would compress these values without revealing additional phases. For these\nreasons, we have retained the linear scaling. Here is a figure where we have put the Y-axis\nas log scale and as you can see, this is not really showing additional interesting data.\nLine 110: mention the temperatures tested in the main text and the temperatures where\ntps2 mutant had defective growth.\nResponse: We thank the reviewer for this suggestion. We have revised the Results section\nto explicitly state the temperatures tested and to highlight the condition where the tps2Δ\nmutant showed defective growth. The revised text now reads (Line 113-117):\n“To assess thermal stress tolerance, growth assays were conducted at 37 °C, 39 °C, and\n42 °C. All mutant strains exhibited varying degrees of growth inhibition, with the tps2Δ\nstrain exhibiting the most severe defect. Specifically, the tps2Δ strain displayed impaired\ngrowth at 39 °C and completely failed to grow at 42 °C, indicating a marked loss of high-\ntemperature tolerance (Figure 1B).\nLine 113: why was the double mutant less affected than the tps2 mutant?\nResponse: On the one hand, trehalose itself is important for thermal stress tolerance, which\nexplains why the tps1Δ and tps1Δ tps2Δ strains exhibited growth defects at elevated\ntemperatures. On the other hand, our data indicate that the pronounced thermosensitivity\nof the tps2Δ mutant is primarily caused by the toxic accumulation of trehalose-6-phosphate\n(T6P) when Tps2 (T6P phosphatase) is absent. In the double mutant (tps1Δtps2Δ), T6P\nsynthesis is abolished due to the loss of Tps1, thereby preventing T6P accumulation and\nalleviating the toxicity observed in tps2Δ, even though trehalose itself is not produced. This\ninterpretation is consistent with our metabolite measurements, which revealed high\nintracellular T6P in tps2Δ but not in the double mutant.\nLine 117: first describe the stresses that were tested and then the results\nResponse: We have revised the text accordingly to first list the stress conditions and then\ndescribe the results. The revised text now reads (Lines 121-129):\nTo evaluate stress resistance, the mutant and WT strains were tested against oxidative stress\n(hydrogen peroxide, H₂O₂), osmotic stress (sodium chloride, NaCl), membrane stress (sodium\ndodecyl sulfate, SDS), and cell wall stress (calcofluor white, CFW, and Congo red, CR). No\nsignificant differences were detected between the wild-type and mutant strains under these\nstress conditions (Figure 1C). In contrast, clear differences emerged under cell wall stress.\nThe tps2Δ strain showed increased sensitivity to CFW, a chitin-binding dye, but exhibited\nenhanced resistance to CR, which binds to β-glucan in the cell wall. By comparison, tps1Δ\nand tps1Δ tps2Δ strains did not display significant differences in sensitivity to either CFW or\nCR relative to the WT strain (Figure 1C).\nLine 132/132 indicate that reduced relative resistance equates to increased susceptibility\nand add……in comparison to WT .\nResponse: We have revised the text to explicitly state that reduced resistance corresponds to\nincreased susceptibility and have added the comparison to the WT strain. The revised text\nnow reads (Line 151-155):\nIn this context, reduced relative resistance is interpreted as increased susceptibility\ncompared with the WT strain, while tolerance refers to the ability of a subpopulation of cells\nto grow slowly but persistently at drug concentrations above the MIC. Together, these assays\nallowed us to evaluate both resistance and tolerance phenotypes in the trehalose-deficient\nmutants.\nLine 137: is involved\nResponse: The wording has been corrected, and the phrase now reads “is involved” in the\nrevised manuscript.\nLine 147 & 161: there are some subtle changes in susceptibility of the tps1 and tps1/tps2\ndouble mutant. Better to state that rather than no notable changes\nResponse: Thank you for the suggestion. While MIC differences versus WT were small in our\nassay (Fig. 2A), the heatmap (Fig. 2B) shows modest shifts in relative resistance for the tps1Δ\nand tps1Δ tps2Δ mutants. We have therefore removed the previous statement of “no\nnotable changes (Line 161)”\nPresume from Figure 5 onwards the analysis was done on the Clade I WT and mutant?\nWould be worth stating that at the start of these experiments.\nResponse: Thank you for the suggestion. Analyses from Figure 5 onward were performed\nusing the Clade I wild-type strain B8441 and its mutants. We have added a clarifying\nsentence at the start of this section (Line 276) and noted it in the figure legends.\nLine 255: interesting finding as the general rule is that elevated chitin leads to CFW\nsensitivity and low chitin (e.g. in chs3 mutant) results in CFW resistance. This finding of low\nchitin and high sensitivity will probably reflect other alterations in the cell wall that impact\non CFW sensitivity.\nResponse: Thank you for raising this important point (see also reply to referee 1). We agree\nthat the phenotype of tps2Δ—lower chitin content yet heightened sensitivity to calcofluor\nwhite (CFW)—does not follow the usual correlation between total chitin levels and CFW\nsusceptibility. Consistent with previous reports 1-3, our data suggest that CFW sensitivity\ndepends on chitin accessibility and overall cell-wall organization, not simply on chitin\nabundance4. We have revised the text to clarify this distinction.\nWe also considered whether loss of the stress-protective function of trehalose could explain\nthe phenotype. However, both tps1Δ and tps1Δ tps2Δ strains, which are similarly defective\nin trehalose biosynthesis, do not display increased CFW sensitivity compared with the wild\ntype (Figure 1C,D). This argues against trehalose depletion as the main cause.\nInstead, we propose that accumulation of trehalose-6-phosphate (T6P) in tps2Δ disrupts\ncell-wall architecture—for example, by altering β-glucan–chitin cross-linking and increasing\nwall porosity—thereby exposing the remaining chitin to CFW and leading to hypersensitivity\ndespite the overall reduction in chitin content.\nLine 253- Perhaps not surprising that you didn’t see changes in CHS3 expression as it is\nmainly regulated post-translationally. It would have been informative to look at changes in\ngene expression in presence of echinocandin stress. Any thoughts on mechanism of down\nregulating CHS1 and CHS2 expression? Related to elevated T6P levels and turgor pressure?\nResponse: Thank you for the helpful comment. We agree that the possible reason of lacking\nof change in CHS3 expression is consistent with its control at the post-translational level\n(trafficking/activation).\nIn WT cells, we do observe increased CHS1, CHS2, and CHS3 gene expression under\nechinocandin stress. In tps2Δ, however, even low doses of caspofungin severely impair\ngrowth and often cause cell death, which prevents reliable measurement of chitin-synthase\ntranscripts during drug exposure.\nTo date, there is little research investigating the function of the C. auris chitin synthases and\ntheir regulation. In S. cerevisiae and C. albicans the class IV enzyme Chs3 produces most cell-\nwall chitin, while Chs1 and Chs2 play more specialized roles (e.g., repair and septum\nformation). However, for C. auris, we cannot definitively explain the mechanism of lower\nCHS1 and CHS2 expression In tps2Δ strain. We propose several, non-exclusive possibilities:\nMetabolic feedback: Accumulated T6P can disturb carbon flux and reduce UDP-GlcNAc\navailability; cells may lower CHS1/CHS2 expression when the substrate is limited.\nOsmotic/turgor effects: Altered trehalose/glycerol balance in the tps2Δ strain could change\nturgor and indirectly reduce septation-linked chitin synthases.\nGFA1 gene product is under allosteric regulation through a negative feedback loop\nregulated by UDP-GlcNAc levels and elevated GFA1 levels may partially circumvent the\nnegative feedback leading to higher UDP-GlcNAc providing more substrate for chitin\nsynthesis. This could be considered in the discussion section.\nResponse: We thank the reviewer for this insightful suggestion. We have now incorporated\nthis point into the Discussion (Line 540). Specifically, we state:\n“Instead, the upregulation of GFA1 in tps2Δ strain likely reflects a negative feedback\nmechanism regulated by intracellular UDP-GlcNAc, whereby elevated GFA1 gene expression\npartially counteracts this feedback to expand the UDP-GlcNAc pool and supply additional\nsubstrate for chitin synthesis.”\nThis addition highlights the potential role of allosteric regulation and negative feedback in\nmodulating UDP-GlcNAc availability and chitin biosynthesis.\nFigure 7A the fructose-6-P to hexosamines is a reversible reaction.\nResponse: We have revised Figure 7A to indicate that the conversion of fructose-6-\nphosphate to hexosamines is a reversible reaction.\nLine 262: glycolysis spelling\nResponse: The spelling of “glycolysis” has been corrected in the revised manuscript.\nVery nice approach to add exogenous GlcN to bypass the requirement for F6P and show the\neffect on drug tolerance, did you test whether this restored chitin levels of the tps2 mutant\ncompletely back to WT.\nResponse: Thanks for this thoughtful comment. We attempted to determine whether\nexogenous glucosamine could restore the resistance and tolerance of the tps2Δ mutant to\ncaspofungin to wild-type levels by supplementing cultures with varying concentrations of\nglucosamine. As shown in our ETEST® and broth dilution assays (Figures 7 and 9), exogenous\nglucosamine partially restored caspofungin resistance and tolerance in the tps2Δ mutant but\ndid not fully return them to WT levels. Higher concentrations of glucosamine also failed to\nachieve complete restoration. For this reason, we did not further measure chitin levels\nunder glucosamine-supplemented conditions.\nIn vivo testing showed no differences in burdens when WT was compared with mutant\n(presuming Clade 1 background) but when mice were treated with caspofungin tps2 mutant\nhad significantly reduced burdens. Was there any difference in survival? Did the untreated\nmice succumb to disease and the caspofungin treated survive? At what time point were the\nfungal burdens assessed.\nResponse: In caspofungin-treated mice, the tps2Δ mutant exhibited a significantly reduced\nfungal burden compared with WT (Figure 10C). However, there were no significant\ndifferences in survival between untreated and caspofungin-treated groups. To further\nillustrate this, we have added a new panel (Figure 10B) showing mouse weight monitoring\nfrom day 0 to day 6 post-infection, which did not reveal differences in health status across\ngroups. Fungal burden was assessed at day 6 post-infection following treatment with either\ncaspofungin or PBS.\nLine 309: you haven’t directly shown that “tolerance” is lost in vivo only that burdens were\nreduced. To show loss of tolerance the cells from infected organs could have been plated on\neTest strip plates.\nResponse: Thanks for this important clarification. We agree that our in vivo experiment\ndemonstrates reduced fungal burden during caspofungin treatment, but does not directly\nassess drug tolerance in vivo. Recovering organ isolates for ex vivo ETEST/time-kill assays\nwould be required to demonstrate loss of tolerance, and archived samples from the\ncompleted study are unavailable. Accordingly, we have removed wording implying an in vivo\nloss of tolerance). Our in vitro assays can only show decreased caspofungin tolerance of\ntps2Δ, which may contribute to the in vivo phenotype; however, we refrain from inferring a\ncausal link and note in the Discussion that future studies will assess tolerance directly from\norgan isolates (although this may also not be straightforward as it requires purification and\nenough cells have to be available to do an Etest).\nLine 323: you haven’t shown directly that UDP-GlcNAc levels are reduced, you have shown\nthat chitin levels are reduced and you should also consider that GFA1 expression is elevated.\nIncidentally increased expression of GFA1 in response to cell wall stress has been previously\npublished in S. cerevisiae (PMID: 11895440) and this could be added to the rationale behind\ninvestigating this gene. The supplementary data on gfa1 mutant may not be best introduced\nin the Discussion section, where it comes as a bit of a surprise, could it be included earlier in\nthe Results?\nResponse: Thank you for these constructive suggestions. We have revised the manuscript\naccordingly:\nUDP-GlcNAc wording. We did not directly quantify UDP-GlcNAc. We therefore softened the\nlanguage to state that chitin content is reduced, consistent with altered flux through the\nUDP-GlcNAc pathway (Line 415).\nPlacement of Gfa1 data. We moved the data of the gfa1 mutant findings from the\nDiscussion to the Results (starting at line 250), and we retain a concise interpretation in the\nDiscussion (line 346).\nFigure 10 spelling chitin synthesis, N of N-acetylglucosamine should be in italics.\nResponse: Thank you for pointing this out. We have corrected the spelling of \"chitin\nsynthesis\" and have italicized the \"N\" in N-acetylglucosamine in Figure 10 (Revised as Figure\n11).\nLine 680: 3 weeks?\nResponse: In our in vivo experiments, we used 7-week-old BALB/c female mice (Janvier Labs)\nMethods: some suppliers are missing: e.g. nourseothricin, anti-β-glucan primary antibody\nFc-Dectin-1, Concanavalin A-Alexa Fluor 488, IgG secondary antibody conjugated to Alexa\nFluor 488\nResponse: We have updated the Methods to include supplier.\nReviewer #3 (Remarks to the Author):\nThis tightly reasoned report presents mechanistic insight into the emerging pathogen\nCandida auris' insensitivity to echinocandin antifungals. Candida auris has become an\nincreasing menace over the last decade due to its resilience to antifungals and disinfectants,\nso on that basis the study is highly significant. Moreover, the findings point to an enzyme\nwith a causal role, and in this way the authors have a druggable target. This element of the\nstudy further increases its significance.\nA second strength of the study is the thoroughness of the analysis. For example, strains\nfrom all four major pathogenic C. auris clades are tested. Also, metabolic inferences are\npursued and validated with metabolomic characterization. Finally, a point that all readers\nwill appreciate is the simplicity of the explanation at which the authors arrive. It is the kind\nof outcome that even students in my Introductory Microbiology class could understand and\nappreciate! In fact, if this work is published in time, I may even include it in a lecture this fall.\nThe one minor experimental criticism that I have is that the most revealing mutant, the\ntps2Δ strain, was not complemented. There was a G418 marker developed by Santana and\nO'Meara in Nat Commun. 2021 Dec 10;12(1):7197 that might be useful, or the authors could\nrecycle their NAT marker as they do for other purposes. I do not doubt the overall\nphenotypic results as they stand - the authors make the mutant in multiple strains, for\nexample - but for some of the findings, such as reduced chitin accumulation and reduced\nhexokinase activity, complementation would ensure that the conclusions reach the highest\nstandard.\nThe one minor manuscript preparation criticism that I have is that the Discussion and\nResults sections seem to overlap more than necessary. I wondered if the authors might\nbenefit from merging the two into a single \"Results and Discussion\" format. This issue is\nmore of a matter of taste though, which is why I consider it quite minor.\nResponse: We appreciate the reviewer’s point regarding the absence of reintegrant controls.\nTo address this, we generated a TPS2-reintegrant strain (tps2Δ+TPS2) by Episomal Plasmid\nmethod 5 and re-evaluated key phenotypes. Complementation restored wild-type behavior\nacross assays: thermotolerance, cell-wall stress responses (CFW and CR), caspofungin\nsusceptibility and tolerance (BDA/Etest) and hexokinase activity assay. These data\ndemonstrate that the phenotypes observed in tps2Δ are specifically attributable to loss of\nTPS2. The new results and figure references have been added to the manuscript (see Fig.\nS1–S5).\nThank you for noting the redundancy. We have streamlined the Discussion and Conclusion\nby removing repetitive descriptions of results and consolidating overlapping passages. The\nrevised sections retain all key findings and interpretations while improving clarity and\nreadability.\nMy one strong recommendation with regard to manuscript preparation is that the authors\nshould define clearly what resistance and tolerance each mean. These are terms with\ndistinctions that can be a little hazy among a broad audience. I am 99% certain that the\nauthors' assays are completely in line with the simple definitions that I always refer to\n(Berman and Krysan, Nat Rev Microbiol. 2020 Jun;18(6):319-331), so I do not question the\nauthors' assays and conclusions; my recommendation is really to make sure the work can be\nappreciated by a broad readership.\nResponse: Thank you for this helpful suggestion. We have added explicit definitions of\nresistance and tolerance (citing Berman & Krysan, Nat Rev Microbiol, 2020) to the\nIntroduction section, and we clarified them in the relevant figure legends (Figure 2)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for the thoughtfulness of their responses, additional experiments and modifications the manuscript. I have\nno remaining concerns.", "reviewer_2": "The authors have addressed the comments of the reviewers, performing new experiments, adding new data and improving\nclarity of the manuscript."}, "author_response": ""}]

Decision letter