Candida auris Metabolism and Growth Preferences in Physiologically Relevant Skin-like Conditions
1Microbial Genomics Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
2Department of Microbiology and Immunology, Georgetown University, Washington D.C., USA
3Department of Nutrition and Food Science, University of Maryland, College Park, MD, USA
* Corresponding author; email: jsegre@nhgri.nih.govABSTRACT
Candida auris is an opportunistic, multidrug-resistant yeast with high capacity of skin colonization in healthcare settings, which can lead to subsequent infections with high mortality rates. Given the recent emergence of at least four distinct clades at the global scale, little remains known about how C. auris is so adept at growing on skin and the key genes and pathways it utilizes to metabolize the scarce nutrients available. Here, we identify the roles that conventional and alternative carbon metabolism genes and metabolic pathways have in facilitating C. auris growth through laboratory-based experiments and bioinformatics analyses. In artificial skin-like media, all four clades of C. auris were more capable of growing than C. albicans SC5314, a clinically relevant counterpart. By investigating the differential regulation of C. auris when growing in skin-like media as compared to rich fungal media, we uncovered hundreds of genes in multiple metabolic pathways. To further test the mechanisms of these metabolic pathways, we deleted several non-essential gene candidates including FOX2 (B9J08_002847), CAT2 (B9J08_000010), and ICL1 (B9J08_003374). The mutant strains all exhibited abrogated growth in skin-like media and demonstrated nutrient preferences that differed from the wild type. Thus, we propose a model of how C. auris has the capacity to metabolize nutrients that are naturally available on skin by changing its metabolic profile. Targeting these metabolic pathways to mitigate C. auris growth on skin is a potential avenue to explore in controlling the spread of this emerging human fungal pathogen.
IMPORTANCE
Candida auris is an emerging fungal pathogen with skin as its primary site of colonization and subsequent transmission. Here, we show the importance of conventional and alternative carbon metabolism for C. auris’ ability to grow in artificial skin-like media. This knowledge provides a better understanding of C. auris metabolism and sheds light on genes and pathways that could be targeted to interfere with persistent skin colonization.
INTRODUCTION
Candida auris is a yeast that was first identified in a clinical report in a Japanese hospital in 2009 and has since independently emerged across four continents (1, 2). Epidemiological and genetic analysis categorized these four clades as: South Asia: Clade I, East Asia: Clade II, South Africa: Clade III, and South America: Clade IV (1). Tens to hundreds of single nucleotide polymorphisms (SNPs) may exist between two isolates of the same clade, but thousands of SNPs distinguish clades. New reports have identified a potential Clade V in Iran (3), and a potential Clade VI in Bangladesh (4) and Singapore (5). The origin of C. auris prior to colonizing humans and its recent emergence remains a topic of deep interest (6). C. auris has emerged as a cause of outbreaks in healthcare settings such as nursing homes and hospitals where it can persist for extended periods of time in rooms with colonized patients on high touch surfaces like mattresses, tables, chairs, floors, and doorknobs (7-10). C. auris exhibits resistance to disinfectants typically used in healthcare settings (11). C. auris can produce invasive candidiasis of the blood which can lead to life-threatening disease for individuals with various risk factors including compromised immunity, diabetes, stroke, indwelling medical devices, long term use of antibiotics or antifungals, and recent surgery (12, 13). The mortality rate for C. auris invasive infections is estimated to be 30-60% (12). A 2020 systematic review and meta-analysis evaluated over 4,700 cases reported in at least 33 countries and showed that C. auris (Clades I-IV) exhibit a high antifungal resistance profile of 91% resistance to fluconazole, 12% resistance to amphotericin B, 12.1% resistance to caspofungin, 0.8% resistance to micafungin, and 1.1% resistance to anidulafungin (13).Taken together, the challenges posed by C. auris contributed to its designation as an Urgent Threat in the CDC’s 2019 Antibiotic Resistance Threats Report in the US (14) and as a Critical Priority in the WHO’s 2022 Fungal Priority Pathogens List (15).
Skin is considered the primary reservoir for C. auris which differs from other Candida species that are more frequently associated with the gastrointestinal, oral, and urinary tracts (16). Vallabhaneni et al., 2017 evaluated the first C. auris infection cases in the USA reported to the CDC and they identified C. auris on human skin in the groin and axilla as well as the nares and rectum of patients (10). Eyre et al., 2018 found C. auris on the axilla and groin of patients in a neurosciences ICU at Oxford University Hospital, UK where an outbreak occurred from 2015-2017 (17). Adams et al., 2018 detected the presence of C. auris on the nares, axilla, and groin of patients in healthcare facilities in New York City, New York, USA in 2017 (7). Sexton et al., 2021 found C. auris in bilateral axillary and inguinal composite skin in a ventilator-capable unit in Chicago, Illinois, USA in 2018 (9). Proctor et al., 2021 screened ten body sites of residents of a skilled nursing facility in Chicago, Illinois over a three month point prevalence survey and found that C. auris prevalently colonized anterior nares, palm and/or fingertips, and toe web (18). Proctor et al. also discovered that residents could be colonized discreetly or simultaneously at multiple skin sites while validating C. auris’ propensity for skin colonization and growth (18). Currently, CDC recommends that healthcare providers screen the axilla and groin to identify C. auris (https://www.cdc.gov/candida-auris/screening/index.html). Taken together, these studies show that C. auris persists on skin which predisposes a patient to infection (16). Moreover, long term skin colonization also favors transmission from patient to patient and overall spread in healthcare settings (19).
Several simulation studies and animal models have demonstrated that C. auris can grow and persist for extensive periods under skin-like conditions. Eix et al., 2022 demonstrated that C. auris could grow in a formulation of synthetic sweat medium for 24 hours and form a biofilm (20). Horton et al., 2020 demonstrated with a pig skin model supplemented with synthetic sweat medium that C. auris forms higher burden biofilms than C. albicans (21). Johnson et al., 2021 showed that C. auris could persist on pig skin for days at a time when grown in synthetic sweat media and that it can grow near the hair follicle (22). Huang et al., 2021 also demonstrated that C. auris could reside in murine skin within the hair follicle/sebaceous gland for months after the skin surface swabs tested negative (23). Guolei et al., 2025 showed that C. auris can coat the murine hair shaft and reside in the hair follicle (24). Santana et al., 2023 discovered an adhesin, SCF1, that is specific to C. auris that is critical for human and mouse skin colonization (25). Shivarathri et al., 2024 also showed that Hog1 mitogen-activated protein kinase is essential for efficient skin colonization (26). Advancing understanding of the genes and metabolic pathways involved in C. auris fitness in these types of scenarios may help facilitate development of more effective strategies for control.
Skin is a nutrient poor environment that is not particularly hospitable to an incoming pathogen (27). Skin also has a range of distinct microenvironments: sebaceous, dry, moist, and foot (27). Each skin microenvironment has its own nutrient profile based on sweat and sebum gland densities which vary depending on body site (27). Sweat glands (eccrine and apocrine) secrete glucose, amino acids, salts, and other nutrients that microorganisms can use to metabolize and grow (28). Sebaceous glands produce triglycerides, free fatty acids, and waxes that also provide additional nutrients for metabolism and growth (28). The ability to metabolize both sweat and sebum nutrients is therefore hypothesized to be key for growth and long-term persistence on skin. An understudied aspect of C. auris research is the metabolic strategies it employs to grow in various sweat and sebum nutrient sources.
Previous work has shown that to metabolize glucose C. albicans uses conventional carbon metabolism pathways such as glycolysis, the citric acid cycle, and the electron transport chain as well as oxidative phosphorylation for growth (29). However, in nutrient poor environments without glucose C. albicans must scavenge and use alternative carbon metabolism (30). For example, C. albicans employs β-oxidation (POX1, FOX2, POT1), the glyoxylate cycle (ICL1, MLS1), and gluconeogenesis to generate glucose from fatty acids for growth (31-36). C. albicans also employs the carnitine shuttle (YAT1, YAT2, CAT2) to assist in transport of acetyl-CoA (29), multiple secreted lipases (LIP) for nutrient acquisition of lipids and adaptation (37, 38), phospholipases and proteases for nutrient acquisition, and amino acid permeases and transporters for additional nutrient uptake (39). Taken together, both conventional and alternative carbon metabolism allow C. albicans to grow in various nutrient sources.
Here, we investigate whether C. auris employs similar metabolic strategies as C. albicans to grow in various nutrient sources like sweat and sebum nutrients. Our work revealed that C. auris uses conventional carbon metabolism enzymes and metabolic pathways to metabolize sweat nutrients like glucose, amino acids, and salts. We show that as those nutrients are depleted, C. auris changes its metabolic profile and uses alternative carbon metabolism pathways to metabolizes sebum nutrients like fatty acids. Collectively, our results show that C. auris shifts its metabolic profile to allow it to grow and persist with limited nutrients in a skin-like environment.
RESULTS
C. auris growth in rich media and skin-like media
Past studies have shown that C. auris is capable of growing in formulations of synthetic sweat medium (20-22). However, no studies have evaluated what genes and metabolic pathways C. auris might use to metabolize synthetic sweat medium nutrients, including the addition of a sebum.
To establish this experimental system, we first sought to assess differential growth patterns of C. auris AR0387 (Clade I), AR0381 (Clade II), AR0383 (Clade III), and AR0385 (Clade IV), and C. albicans SC5314 in nutrient-rich media (YPD) as well as artificial skin-like media supplemented with sebum (Sweat + 0.1% Sebum) (Fig. 1A). In YPD, the area under the curve (AUC) for C. albicans compared to each of the C. auris clades was significantly higher indicating more robust growth in nutrient-rich conditions (P < 0.05) (Fig. 1B). In contrast, all C. auris clades had significantly greater AUC than C. albicans in Sweat + 0.1% Sebum (P < 0.001) (Fig. 1B). Thus, C. auris grew better than C. albicans in the media simulating skin-like environmental conditions.
Then we sought to understand what genes C. auris and C. albicans might be using to grow at logarithmic growth phase (8 hr.) in Sweat + 0.1% Sebum compared to YPD using RNA-sequencing. The principal component (PC) analysis (PCA) plot showed that C. albicans shifted along PC1 between the different media, while all four clades of C. auris shifted along PC2 between the media (Fig. 1C). We also compared growth in the two media types and discovered 620 genes were significantly upregulated by C. auris in Sweat + 0.1% Sebum compared to 490 genes significantly upregulated in YPD (Fig. 1D). KEGG pathway analysis also revealed that the genes upregulated in Sweat + 0.1% Sebum were most frequently involved in pathways related to metabolism, biosynthesis of secondary metabolites, peroxisomes, carbon metabolism, and oxidative phosphorylation.
Growth analysis of C. auris strains with deletion of individual lipid metabolism genes
To test our functional hypothesis, we targeted the deletion of differentially expressed genes to assess whether they would provide selective growth defect in Sweat + 0.1% rather than YPD. In our RNA-sequencing analysis we noticed that C. auris was upregulating 32 lipid metabolism genes in Sweat + 0.1% Sebum compared to YPD. To interrogate if these genes were key for C. auris growth in Sweat + 0.1% Sebum, we made individual mutant strains with Fusion PCR by replacing the genes with a drug resistance marker (40). The genes of interest were LIP1 (B9J08_004173), LIP2 (B9J08_004176), FOX2 (B9J08_002847), CAT2 (B9J08_000010), and ICL1 (B9J08_003374). We were specifically interested in the LIP genes, because of their ability to metabolize triglycerides (38). We were interested in FOX2 because of its role in alternative carbon metabolism to metabolize fatty acids through β-oxidation (41). We were interested in CAT2 because of its ability to assist in transport and break down of acetyl-Coa (29). Lastly, we were interested in ICL1 because of its role in alternative carbon metabolism to generate citric acid cycle intermediates through the glyoxylate cycle (32-36).
The lip1Δ, lip2Δ, fox2Δ, cat2Δ, and icl1Δ mutants did not show a growth defect in the nutrient-rich YPD compared to C. auris WT and the AUC was not significantly different between any of the strains (P > 0.05) (Fig. 2A). For the Sweat + 0.1% Sebum curves, the LIP mutants grew equally as well as C. auris WT (Fig. 2A). However, fox2Δ, cat2Δ, and icl1Δ had notable growth defects in Sweat + 0.1% Sebum and the AUC was significantly lower than C. auris WT (P < 0.01) (Fig. 2A). Collectively these results indicate that fox2Δ, cat2Δ, and icl1Δ had significantly abrogated growth in Sweat + 0.1% Sebum media, but not in YPD media.
Next, to complement the in vitro broth media assessment, we assessed if growth defects were observed on solid agar. Again, no growth defects were observed on YPD agar (Fig. 2B). However, on the Sweat + 0.1% Sebum agar growth was observed to be abrogated for fox2Δ, cat2Δ, and icl1Δ (Fig. 2B). To assess the growth capacity of C. auris WT and fox2Δ, cat2Δ, and icl1Δ we then performed a serial dilution spot assay onto YPD or Sweat + 0.1% Sebum agar. Again, growth defects were observed specifically on Sweat + 0.1% Sebum (Fig. 2C). Collectively, fox2Δ, cat2Δ, and icl1Δ displayed abrogated growth defects on Sweat + 0.1% Sebum agar, but not YPD agar.
Sweat and sebum gradient assay plates demonstrate nutrient preferences between C. auris WT and mutant strains
We next assayed whether different growth phenotypes were observable for C. auris WT and the mutant strains when sweat and sebum nutrients were varied. To accomplish this, a two-dimensional gradient assay plate was used to grow wild type and mutant strains at eight concentrations of sweat (0.03X to 4X) and sebum (0.002% to 0.25%) for a total of sixty-four growth conditions. Each gradient assay plate was inoculated and assessed for growth at 24 and 48 hours. Data were normalized and optical density was plotted as a heatmap to show differences in nutrient preferences (Fig. 3A).
Next, a multiple linear regression analysis was performed for C. auris WT and the mutant strains to determine nutrient preferences for growth in the gradient assay plate over time. C. auris WT had a sebum preference over time. Fox2Δ had a slight sebum preference at 24 hours, but lost preferences over time likely due to the inability to metabolize sebum nutrients without FOX2. Cat2Δ also had a slight sebum preference at 24 hours, but developed a stronger sebum preference over time likely because it can still metabolize sebum nutrients without CAT2. Icl1Δ had a slight sebum preference over time, but most likely does not gain a strong sebum preference due to the inability to adequately metabolize sebum nutrients without ICL1. Collectively, these results indicate that fox2Δ and icl1Δ had the most distinct nutrient preferences, whereas cat2Δ behaved more similarly to C. auris WT (Fig. 3B; Fig. 3C).
RNA-sequencing analysis of C. auris WT compared to mutant strains
Next, we wanted to explore the genetic pathways altered within fox2Δ, cat2Δ, and icl1Δ that accompany the previously observed growth defects. To accomplish this, we assessed differences in gene expression at logarithmic growth phase (8 hr.) in Sweat + 0.1% Sebum by comparing C. auris WT and fox2Δ, cat2Δ, and icl1Δ with RNA-sequencing analysis.
Volcano plots were generated to identify the most differentially expressed genes between C. auris WT and fox2Δ, cat2Δ, and icl1Δ with a log2 fold change > 1 and adjusted P-value < 0.05. (Fig. 4A). As a control, and reassuringly, the most differentially expressed gene for each comparison was the gene removed from the genome in each mutant strain. When grown in Sweat + 0.1% Sebum, fox2Δ upregulated 184 genes, cat2Δ upregulated 40 genes, and icl1Δ upregulated 67 genes compared to the wild type. Sixteen genes were commonly upregulated and 3 genes were commonly downregulated by all three mutants in comparison to C. auris WT (Fig. 4B). Of the 16 commonly upregulated genes, 7 were annotated: ILV3, LEU1, MET16, PRX1, ERG24, ARG3, and DAG7. Of the 3 commonly downregulated genes 2 were annotated: WOR4 and AQY1. KEGG pathway analysis revealed that the upregulated genes were primarily involved in biosynthesis of secondary metabolites and amino acid synthesis whereas the downregulated genes did not have sufficient information for analysis.
Pathway analysis was also done to evaluate which pathways were upregulated and downregulated comparing each mutant to C. auris WT (Fig. 4C). For fox2Δ, we observed increased expression of tyrosine metabolism, pyruvate metabolism, and glycolysis and gluconeogenesis genes (Fig. 4C). For fox2Δ, we also observed decreased expression of valine, leucine, and isoleucine degradation and biosynthesis, sulfur cycle, propanoate metabolism, peroxisome, lysine biosynthesis, glyoxylate and dicarboxylate metabolism, fatty acid metabolism and degradation, citrate cycle (TCA cycle), carbon metabolism, biosynthesis of unsaturated fatty acids and secondary metabolites, biosynthesis of amino acids, beta-Alanine metabolism, and 2-oxocarboxylic acid metabolism. These results suggest that when FOX2 is deleted the cell responds by increasing the expression of multiple pathways to metabolize available sweat nutrients to account for its hindered ability to metabolize sebum nutrients.
For cat2Δ, we observed decreased expression of valine, leucine, and isoleucine biosynthesis, biosynthesis of amino acids, and 2-oxocarboxylic acid metabolism (Fig. 4C). These results suggest that when CAT2 is deleted it does not affect gene expression as significantly and metabolism is not as altered.
For icl1Δ, we observed increased expression of pyruvate metabolism, glyoxylate and dicarboxylate metabolism, and carbon metabolism. Icl1Δ also had decreased expression of oxidative phosphorylation (Fig. 4C). These results suggest that like FOX2, when ICL1 is deleted the cell responded by increasing the expression of multiple pathways to metabolize available sweat nutrients to account for its hindered ability to metabolize sebum nutrients.
Collectively, these results suggest that FOX2, CAT2, and ICL1 are important for growth in Sweat and Sebum. Moreover, upregulation of additional genes and pathways sheds light on how metabolism occurs in a variable sweat and sebum environment.
DISCUSSION
C. auris is a fungal pathogen of increasingly urgent health risk due to multidrug-resistance and its ability to cause hard to treat bloodstream infections. However, skin is the primary site of C. auris colonization and this predisposes patients to shedding of the organism which promotes seeding of the environment and transmission. Understanding how C. auris can grow on skin and what nutrients it might be utilizing to do so is a significant gap in knowledge regarding this pathogen. Our data reveal that encoding diverse conventional and alternative carbon metabolism genes facilitates growth and nutrient utilization in a skin-like environment.
Skin is a nutritionally diverse location and has four distinct microenvironments (sebaceous, dry, moist, and foot) with varied density of sweat and sebaceous glands depending on body site (27). Sweat glands provide glucose, amino acids, salts, and other nutrients and sebaceous glands provide triglycerides, fatty acids, and waxes (28). However, these nutrients are not abundant on skin, so microorganisms that grow there are generally adapted to this low nutrient environment.
The healthy skin mycobiome, or the collection of fungi on skin, is dominated by Malassezia species (42, 43). Malassezia can grow across the entire body surface, however they are lipophilic, or “fat-loving”, and prefer the lipid-rich environment of the hair follicles/sebaceous gland where there is sebum (43). This is because Malassezia lack fatty acid synthase and cannot synthesize fatty acids on their own, so they scavenge them from their environment (44) Consequently, Malassezia have lipases, phospholipases, and sphingomyelinases (45-47) to break down extracellular lipids from sebum to fatty acids, allowing them to grow (30, 43). Malassezia also lack carbon metabolism genes which is likely an evolutionary consequence of their long-term colonization of the sebum rich areas of skin (44).
C. albicans, which is an ascomycete genetically more similar to C. auris than Malassezia species, can grow on skin but has a greater tropism for the gastrointestinal flora, oral cavity, and reproductive tract. C. albicans also differs from Malassezia because it employs its own set of strategies for metabolism in various nutritional environments. For example, if glucose, the preferred carbon source, is present it will be metabolized with conventional carbon metabolism pathways such as glycolysis to form acetyl-CoA. Then acetyl-CoA will enter the citric acid cycle, a conventional carbon metabolism pathway, to produce eight intermediates and multiple coenzymes. Two coenzymes, NADH and FADH2, enter the electron transport chain and generate ATP through oxidative phosphorylation allowing cellular growth (33). If glucose is unavailable or in low quantities, C. albicans uses alternative carbon metabolism pathways to grow instead. One such pathway is β-oxidation, which is carried out by three enzymes (POX1, FOX2, POT1) in four metabolic steps (29). In β-oxidation, fatty acids are broken down into acetyl-CoA (29). Then acetyl-CoA can enter the glyoxylate cycle, another alternative carbon metabolism pathway, which is unique to Candida species, bacteria, and plants (33). In this process, isocitrate is hydrolyzed to glyoxylate by ICL1. Then acetyl-CoA, from β-oxidation, is condensed with the previously produced glyoxylate to produce malate by MLS1. Malate, a citric acid cycle intermediate, is converted to oxaloacetate, then citrate, and back to isocitrate. This process replenishes intermediates of the citric acid cycle allowing it to function when nutrients are low.
Additionally, the oxaloacetate from the glyoxylate cycle can enter gluconeogenesis, another alternative carbon metabolism pathway, to produce glucose which provides energy to sustain the cell. C. albicans also uses the carnitine shuttle (YAT1, YAT2, CAT2) to transport acetyl-CoA into the mitochondria and peroxisome for downstream metabolism (29). C. albicans also has many secreted lipases (LIP) at its disposal for additional metabolism of lipids too (37, 38). Overall, C. albicans has many strategies it can use to metabolize nutrients in either rich or poor environments to grow with conventional or alternative carbon metabolism respectively.
Other Candida species also use alternative carbon metabolism when glucose is not available. For example, Candida lusitaniae uses β-oxidation (FOX2) and the glyoxylate cycle (ICL1) to metabolize fatty acids as a carbon source to grow and transports acetyl-units with carnitine acetyl-transferase systems (CAT2) (48). Candida tropicalis uses β-oxidation (FOX2) to break down short length fatty acids to generate acetyl-CoA and then transports it to the mitochondria and peroxisome via carnitine acetyl-transferase systems (CAT2) (49). Candida glabrata uses β-oxidation (FOX2) to generate acetyl-CoA, carnitine acetyl-transferase systems (CAT2) to move acetyl-CoA, and the glyoxylate cycle (ICL1) to process acetyl-CoA (50).
Prior to this study, there has been limited knowledge about what metabolic pathways C. auris might use to grow in a skin-like environment. Here, we showed that C. auris uses lipid metabolism genes to grow in Sweat + 0.1% Sebum media, but those genes are not essential for growth in glucose rich YPD. We found that key genes that facilitate this growth are FOX2, CAT2, and ICL1, similar to other Candida species.
FOX2 is important for C. auris because it drives the alternative carbon metabolism pathway β-oxidation, which metabolizes fatty acids to acetyl-CoA (Fig. 5A; Fig. 5B; Fig. 5C). Major nutrients of our Sweat + 0.1% Sebum media are fatty acids, not glucose. Therefore, if FOX2 cannot function, then fatty acids will not be properly metabolized, and the cell will not grow. Similarly, if CAT2 cannot function then acetyl-CoA will not be properly transported to the mitochondria or peroxisome, downstream metabolic pathways will not function, and the cell will not grow (Fig. 5A; Fig. 5C). Lastly, if ICL1 cannot function then the glyoxylate cycle will not produce citric acid cycle intermediates and coenzymes properly and the cell will not grow (Fig. 5A. Fig. 5C). This corelates with past studies where a C. albicans FOX2 mutant (51), an ICL1 mutant (32), and a CAT2 mutant (52) did not grow on oleic acid as a nutrient source.
We were also interested to learn if C. auris had growth preferences in Sweat + 0.1% Sebum. When we grew C. auris in a gradient assay plate with a broad range of nutrients we noticed that it could grow in the sweat nutrients (glucose), but it preferred the sebum nutrients (fatty acids) (Fig. 3). This was noteworthy because it indicated that C. auris metabolized the sweat nutrients with conventional carbon metabolism first, and then the sebum nutrients with alternative carbon metabolism second. This long-term sebum preference is similar to skin commensal Malassezia species which are lipophilic, or ‘fat-loving’, and prefer the lipid-rich environment of the hair follicles/sebaceous gland (43). This could explain why C. auris eventually navigates into the pilosebaceous unit where there is available sebum (22, 23). Perhaps, C. auris is also a ‘fat-loving’ microbe too which could explain why it can grow and persist on skin so effectively. Moreover, C. auris may persist in this niche unlike other pathogens because the available fatty acids aren’t antimicrobial, but rather a source of nutrition (44). C. auris may perhaps outcompete Malassezia in this niche. Fox2Δ did not metabolize the range of available sebum nutrients like the wild type validating its importance in fatty acid metabolism. Curiously, cat2Δ behaved more similarly to the wild type. Perhaps this is because the carnitine shuttle is not essential for alternative carbon metabolism, and C. auris might have other ways to move acetyl units into the organelles like C. albicans (29). Icl1Δ never developed a strong sebum preference relative to the wild type. This also indicated ICL1’s importance in fatty acid metabolism.
We also discovered that fox2Δ upregulated genes in glycolysis/gluconeogenesis and pyruvate metabolism pathways compared to the wild type in Sweat + 0.1% Sebum (Fig. 4C). We hypothesize this is occurring because fox2Δ is trying to metabolize the glucose and other sweat nutrients, but not the fatty acids in sebum, since it is unable to do so without the gene. Fox2Δ downregulated sixteen metabolic pathways (Fig. 4C).
We hypothesize this is occurring because FOX2 is a key alternative carbon metabolism gene and without it downstream metabolism is significantly altered in many pathways. Cat2Δ only downregulated three metabolic pathways (Fig. 4C). We hypothesize this is occurring because CAT2 is not as directly involved in alternative carbon metabolism. Like fox2Δ, icl1Δ upregulated pyruvate metabolism as well as glyoxylate, dicarboxylate, and carbon metabolism (Fig. 4C). We hypothesize this is occurring because icl1Δ is also trying to metabolize glucose from sweat nutrients because it cannot metabolize the fatty acids in sebum (Fig. 4C). Collectively, these results suggest that FOX2 and ICL1 are important for growth in Sweat + 0.1% Sebum media with CAT2 playing a minor role.
We also discovered 16 commonly upregulated and 3 commonly downregulated genes for the mutants compared to the wild type (Fig. 4B). Of the 16 commonly upregulated genes, 7 had annotations. ILV3, LEU1, MET16, and ARG3 were involved in amino acid biosynthesis. PRX1 is involved with reducing hydrogen peroxide to water, DAG7 is a possible secretory protein, and ERG24 is involved with ergosterol biosynthesis. Perhaps the mutants are upregulating multiple amino acid biosynthesis genes because the amino acids they synthesize (valine, isoleucine, leucine, sulfur amino acids, and arginine) are important for proper metabolism in a low nutrient environment like Sweat + 0.1% Sebum. PRX1 may be upregulated to mitigate hydrogen peroxide production during metabolism. Lastly, ERG24 may be upregulated for proper synthesis of ergosterol in Sweat + 0.1% Sebum. Of this group of genes, it is possible that ERG24 could be a potential drug target as C. albicans mutants of this gene are susceptible to allylamine antifungals (terbinafine) as well as cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A (53). Of the 3 commonly downregulated genes 2 were annotated. These included WOR4: has domain(s) with predicted role in regulation of DNA-templated transcription and AQY1: Ortholog(s) have water channel activity. It is unclear why these genes were commonly downregulated as they were not related to metabolism. A limitation of this study was that the additional genes discovered could not be analyzed because of insufficient annotations in the KEGG pathway database. More work will be done in the future to understand why the additional differentially expressed genes may be relevant to sweat and sebum metabolism.
Overall, we showed that C. auris has more robust growth than C. albicans in Sweat + 0.1% Sebum media. We also showed that conventional carbon metabolism facilitates growth in sweat nutrients when glucose is present. Alternative carbon metabolism facilitates growth in sebum when fatty acids are present requiring genes like FOX2, CAT2, and ICL1. Additionally, we found more genes and metabolic pathways to explore to better understand C. auris growth capabilities. We hope these efforts will pave the way for better understanding of C. auris metabolism and advance understanding of the genes and pathways that could serve as potential targets for control. More effective strategies are needed to mitigate consequences of C. auris on skin to mitigate risks for transmission and development of bloodstream infection.
MATERIALS AND METHODS
STRAINS
The C. auris and C. albicans strains used in this study are listed in Table S1 in the supplemental material.
MEDIA AND GROWTH CONDITIONS
Strains were grown and plated in two different nutrient sources. The first nutrient source was YPD (Yeast extract peptone dextrose) Broth (Sigma-Aldrich) and YPD Broth + Agar (Sigma-Aldrich) (2%). The second nutrient source was Sweat + 0.1% Sebum with commercial Sweat + 0.1% Sebum used to collect initial data in (Fig. 1). This formulation consisted of three components: Artificial Eccrine Perspiration (Pickering Laboratories, Cat number: 1700-0023), Artificial Sebum (Pickering Laboratories, Cat number: 1700-0700), and 1% Tween 80 (MP Biomedicals, Cat number: 103170). The second Sweat + 0.1% Sebum formulation was a defined media used to collect data in (Fig. 2; Fig. 3; Fig. 4) adopted from Swaney et al., 2023 (27). This formulation consisted of three components: Basal media, Artificial Sweat, and Artificial Sebum. The contents of the media can be found in Table S2. Sweat + 0.1% Sebum agar plates were made by adding 1.5% agar to the Sweat + 0.1% Sebum media previously described.
GROWTH CURVES AND GROWTH METRIC ANALYSIS
Pure culture isolates of the C. albicans and C. auris wild type and mutant strains were grown initially at 34ºC, 200 RPM, overnight in YPD or Sweat + 0.1% Sebum. After normalizing the initial inoculum to OD600 0.1, 10 μl of the freshly washed cells were added to 190 μl of media in a 96-well plate (Nunc™ Edge™ 96-Well, Non-Treated, Flat-Bottom Microplate (Thermo Fisher, Cat: 267544). The plates were placed in a plate reader (Agilent BioTek Epoch 2 Microplate Spectrophotometer, Fisher Scientific) at 34ºC, 200 RPM, for 24 hours. The Growthcurver script was adapted from Sprouffske and Wagner 2016 (54) to plot growth curves and calculate growth metrics such as AUC (area under the curve), k (carrying capacity), r (growth rate), and t_mid (inflection point of the curve). Statistics were calculated using one-way ANOVA with Tukey’s post-hoc tests in R.
GENE DELETION STRATEGY
Mutant strains were generated from C. auris AR0387 with the Fusion PCR protocol and were confirmed with the Colony PCR protocol adopted from Schwarzmuller et al., 2014 (40). Sequencing primers were also generated upstream of the gene of interest that was deleted, in the middle of the selectable marker, and at the 3’ end of the selectable marker to ensure proper deletion of the gene. All primers are available in Table S3.
SWAB AND SERIAL DILUTION PLATES
C. auris AR0387 and mutant strains were grown at 34ºC, 200 RPM, overnight in YPD or Sweat + 0.1% Sebum. After normalizing the initial inoculum to OD600 0.1, swabs were inoculated and streaked on corresponding YPD agar or Sweat + 0.1% Sebum agar, incubated at 34ºC for 24 hours, and imaged.
Then the normalized inoculum was serial diluted 10-fold, twelve times into a 96-well plate. Then a replicator (Boekel, 96-Pin Microplate Replicator, 140500) was used to transfer 1 μl of cells to a corresponding YPD or Sweat + 0.1% Sebum agar plate, incubated at 34ºC for 24 hours, and imaged.
GRADIENT ASSAY PLATES
A gradient assay plate of sweat and sebum was generated which was adopted from Swaney, et al., 2023 (27). In this plate, there are eight concentrations of sweat (0.03X, 0.06X, 0.13X, 0.25X, 0.5X, 1X, 2X, and 4X) and eight concentrations of sebum (0.002%, 0.004%, 0.008%, 0.016%, 0.031%, 0.063%, 0.125%, 0.25%) for a total of sixty-four growth conditions. The remaining wells of the plate were used as controls. Column 9 was the last sweat dilution, column 10 was the last sebum dilution, column 11 was basal media, and column 12 was YPD. Well H12 was also inoculated with C. auris AR0387 as a positive control. Gradient assay plates were grown at 34ºC, 200 RPM. OD600 values were collected with a plate reader at 0 hours, 24 hours, and 48 hours.
Then, the log2 of the sweat and sebum concentrations were used as feature X and the OD600 was used at feature Y. Then a linear regression was trained for each strain. A slope was generated from the X and Y coefficients at each time point to determine how growth changed in sweat and sebum.
Slope thresholds were used to generate nutrient preferences. High sweat = 0° to 60°, Low sweat = 60° to 120°, and No sweat = 120° to 180°. High sebum = 60°-120°, Low sebum = 30° to 60° and 120° to 150°, and No Sebum = 0° to 30° and 150° to 180°.
RNA SEQUENCING AND ANALYSIS
C. albicans and C. auris wild type strains and mutant strains were grown at 34ºC, 200 RPM overnight for 8 and 15 hours in 5 ml of YPD or Sweat + 0.1% Sebum in triplicate. RNA-sequencing reads were mapped using STAR v2.7.11b (55) to the C. albicans SC5314 genome (GCF_000182965.3) from NCBI RefSeq. HOMER v5.1 (56) was used to create tag directories based on mapped data with the script “makeTagDirectory - format sam -checkGC -sspe”, and to calculate raw counts and TPM (transcripts per million) values using “analyzeRepeats.pl rna -count exons”. Differentially expressed genes were identified using HOMER script “getDiffExpression.pl -AvsA -repeats”, which implements DESeq2 (57), with significance thresholds of log2 fold change > 1 and adjusted P value < 0.05. Volcano plots and heat maps were generated to visualize expression difference between C. auris wild type and mutant strains. KEGG pathway enrichment analysis was performed on differentially expressed genes using R package ClusterProfiler v4.10.1 (58) with an adjusted P value threshold of < 0.05.
STATISTICAL ANALYSIS
All experiments were performed with at least three biological replicates as indicated in the figure legends. Analyses were conducted with R and python. Statistics were calculated using one-way ANOVA with Tukey’s post-hoc tests in R. P values < 0.05 were considered statistically significant.
ACKNOWLEDGMENTS
We thank the members of our laboratory for critical discussion on experimental design, execution, and analysis. We thank Lukian Robert for assistance in screening mutants, Milan Stolpman for assistance in gradient assay plates set ups, and Diana Proctor for bioinformatic analysis. We thank the Karl Kuchler Lab for providing the pTS50 plasmid used in the Gene Deletion Strategy. We thank Teresa O’Meara’s laboratory for thoughtful discussion on our experimental design and suggestions for the manuscript. We thank the NIH Intramural Sequencing Center (NISC) for sequencing isolates. We thank the Microarray and Single Cell Genomics Core at NIH for quantification of RNA. This study utilized the computational resources of the NIH HPC Biowulf Cluster (http://hpc.nih.gov). J.A.S. is an Associate Fellow of the Canadien Institute for Advanced Research (CIFAR) program Fungal Kingdom: Threats & Opportunities.
FUNDING
This work was supported by the Division of Intramural Research of the National Human Genome Research Institute (NHGRI).
DATA AVAILABILITY
NCBI BioProject to house all the data
. BioSamples for the WT RNAseq.
BioSamples for the mutant RNAseq.
ETHICS APPROVAL
ADDITIONAL FILES
SUPPLEMENTAL MATERIAL
Supplemental figures:
Table S1. Strains used and designed in this study.
Table S2. Artificial Sweat, Artificial Sebum, and Basal Media recipes used in this study. Table S3. Primers used in this study.
Figure S1. Volcano plots of gene expression between AR0387 and mutant stains (15 hr.)
Figure S2. Heatmap of AR0387 and mutant strains gene expression in Sweat + 0.1% Sebum using the Z score normalized data (15 hr.)
Figure S3. Pathway analysis of AR0387 and mutant strains gene expression in Sweat + 0.1% Sebum (15 hr.)