Host albumin redirects Candida albicans metabolism to engage an alternative pathogenicity pathway

Abstract

Pathogenicity mechanisms of the yeast Candida albicans involve filamentous growth, adhesion, invasion, and toxin production. Interestingly, clinical isolates, and other Candida spp., can cause infection independent of filamentation or toxin production. These strains and species often are characterized as avirulent ex vivo, yet this does not correlate with their potential to cause infection. We hypothesized that specific host factors, which trigger pathogenicity in vivo, are absent in in vitro infection models and thereby clinical isolates can seem avirulent ex vivo. We investigated how albumin, the most abundant protein in humans, impacts infection and cytotoxic potential of C. albicans in vitro. The presence of albumin induces otherwise non-damaging and non-filamentous clinical isolates to cause host cell cytotoxicity. Moreover, avirulent deletion mutants deficient in filamentation, adhesion, or toxin production are restored in their cytotoxicity by albumin. This involves transcriptional and metabolic reprogramming of C. albicans, increasing biofilm formation and production of the oxylipin 13-hydroxyoctadecadienoic acid, driving host cell cytotoxicity. Collectively, our study uncoveres a pathogenicity mechanism by which C. albicans causes epithelial cytotoxicity independent of its conventional virulence mechanisms. This alternative pathogenicity strategy helps to explain the avirulence of clinical isolates ex vivo, when they are separated from the host environment.

Candida albicans normally relies on specific pathogenicity mechanisms to cause tissue damage. This study reveals that when sensing host albumin, C. albicans, even avirulent strains, can trigger an alternative pathogenicity pathway via transcriptional and metabolic reprogramming.

Introduction

Microbial pathogens evolved an impressive arsenal of pathogenicity mechanisms that enable them to cause infections when the circumstances allow it. The opportunistic pathogenic yeast Candida albicans, which colonizes most of the human population, is a prime example¹. When the protective microbiota and immunity are compromised, the opportunity arises that C. albicans can cause infection by relying on key pathogenicity mechanisms². The dogma dictates that the ability to adhere to mucosal surfaces, the formation of hyphae, and the production of the pore-forming toxin candidalysin are essential for C. albicans pathogenicity³.

Typically, C. albicans strains lacking these pathogenicity mechanisms are less or non-pathogenic towards host cells in vitro^{4, 5, 6, 7–8}. It is striking that isolates from candidiasis patients do not always inflict damage to host cells when tested in in vitro infection models⁹. Thus, their capacity to cause infection is uncoupled from the virulence observed ex vivo. Proving this principle, an eed1-deletion mutant, which reverts to yeast growth after inducing germination, displayed avirulence in in vitro infection models⁸. Contrastingly, when mice were infected intravenously, this mutant exhibited similar virulence as the wild type (WT)¹⁰. This suggests that virulence not necessarily depends on filamentation, and that in vitro models lack specifics that enable C. albicans isolates to recapitulate their virulence potential ex vivo. Specific host proteins, absent in vitro, may restore or enhance pathogenicity when re-introduced. In line with this, we previously found that host α1-antitrypsin can augment C. albicans filamentation¹¹. Nakaseomyces glabratus (C. glabrata) is the second most common cause of candidiasis^12,13, yet often it does not cause cytotoxicity in vitro. Albumin, an abundant host protein^{14, 15–16}, activated the pathogenicity of C. glabrata in an in vitro infection model¹⁷. There is evidence that C. albicans can exploit albumin for iron acquisition¹⁸. In contrast, using the highly pathogenic C. albicans strain SC5314, albumin was found to reduce tissue damage through neutralizing the toxin candidalysin, despite augmenting fungal growth¹⁹. However, it remains unresolved how less virulent C. albicans isolates interact with albumin.

Here we investigated how albumin impacts the pathogenicity of several clinical C. albicans isolates and deletion mutants that are commonly considered less- or non-virulent in in vitro infection models. We systematically assessed how albumin causes transcriptional and metabolic reprogramming of C. albicans and unravel how it drives an alternative pathogenicity mechanism.

Results

Clinical strains of C. albicans display different morphology and virulence

The WT reference strain SC5314 is used throughout laboratories worldwide to study C. albicans pathogenicity mechanisms. It is well characterized in terms of its robust filamentation^20,21 and the damage it can inflict to human tissues in in vitro infection models³. Yet, comparing various clinical C. albicans isolates, revealed extreme variation in morphology when grown in vitro (Fig. 1A). Some isolates showed potent filamentous growth resembling SC5314 (C226, C127 and C128), while some switched back to yeast growth after several hours of filamentous growth (C127 and 101²²). Other strains grew as a mixture of hyphal and pseudohyphal morphologies. Despite these being isolates from patients, only a few strains caused cytotoxicity, reflected in release of the cytoplasmic enzyme lactate dehydrogenase (LDH), of A-431 vulvar epithelial cells in vitro (Fig. 1B). SC5314 displayed the strongest cytotoxic potential, followed by C128 and C127, underscoring previous observations that filamentation is essential for C. albicans to cause cytotoxicity to host cells^2,3,7. However, despite hyphal growth, the strain C266 showed no cytotoxicity.

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Fig. 1

C. albicans displays enhanced pathogenic potential in the presence of albumin.

A Morphological differences between clinical C. albicans isolates grown in RPMI-1640 at 37 °C in the absence of host cells. Images were taken after 24 h of incubation at ×20 magnification (scale bar = 100 µm). B Clinical C. albicans isolates grouped according to their cytotoxicity on A-431 cells after 18 and 45 h post infection (hpi). C Cytotoxicity of A-431 cells infected by clinical C. albicans isolates with and without albumin after 18 hpi (top panel) and 45 hpi (bottom panel) B, C: uninfected n = 7 for 18 hpi and n = 6 for 45 hpi; SC5314 n = 6 for 18 hpi and 45 hpi; C128, C127, C227, UC820, C50, C274, and C226 n = 4 for 18 hpi and n = 3 for 45 hpi; 101 n = 3 for 18 hpi and 45 hpi). D Cytotoxicity of HEK293A cells infected by clinical C. albicans isolates with and without albumin after 18 hpi (top panel) and 45 hpi (bottom panel) (n = 6 for 18 hpi; uninfected and SC5314 n = 4 for 45 hpi; C128, C127, C227, C50, C274, C226, and 101 n = 3 for 45 hpi). E Cytotoxicity of A-431 cells infected by C. albicans deletion mutants, attenuated in their virulence potential, with and without albumin (uninfected and BWP17/Clp30 n = 8 for 18 hpi and 45 hpi; efg1ΔΔ/cph1ΔΔ, ece1ΔΔ, als3ΔΔ, and bud2ΔΔ n = 4 for 18 hpi and 45 hpi, and eed1ΔΔ n = 5 for 18 hpi and 45 hpi). Cytotoxicity was quantified by measuring the LDH activity in the supernatant, bars represent the mean ± SEM, and dots represent the mean of the technical replicates of the individual experiments (B–E). Data were tested for significance using one-way ANOVA with Holm-Šídák multiple comparisons tests (B) or two-way ANOVA with Holm-Šídák multiple comparisons test (C– E). P values are provided in the figure for significant comparisons. Source data are provided as a Source Data file.

Albumin increases the epithelial cell damage of non-damaging C. albicans strains

The abundance of albumin throughout different host niches where C. albicans causes infections implies an interaction of the fungus with this protein. Therefore, albumin was introduced into our in vitro infection model to investigate its impact on C. albicans strains with varying pathogenicity. Addition of human albumin potentiated the cytotoxicity of all clinical strains following a prolonged infection (Fig. 1C, Supplementary Fig. 1A). Albumin increased fungal cytotoxicity, particularly of otherwise low damaging clinical isolates, whereas results were less consistent with SC5314, likely due to albumin neutralizing candidalysin¹⁹ of this highly efficient toxin producing strain³. Accordingly, on human embryonic kidney cells (HEK293A) albumin significantly reduced the cytotoxicity induced by the strains SC5314, C128 and C127 at 18 h post infection (hpi) (Fig. 1D). Like on A-431 cells, prolonged infection in the presence of albumin was associated with a significant enhancement of cytotoxic potential of the otherwise low damaging strains UC820, C226, and 101 (Fig. 1D).

As albumin particularly enhanced the cytotoxic potential of C. albicans strains that otherwise were low-damaging, we assessed C. albicans mutants deficient in filamentation (efg1ΔΔ/cph1ΔΔ and eed1ΔΔ), the adhesin and invasin Als3 (als3ΔΔ), the toxin candidalysin (ece1ΔΔ), or thigmotropism (bud2ΔΔ). These mutants lacking crucial virulence genes all increased in their cytotoxic potential following the introduction of albumin in the infection model (Fig. 1E).

We could rule out that the sole addition of any protein at physiological albumin concentrations was sufficient to enhance C. albicans induced cytotoxicity, since other host proteins did not enhance cytotoxicity of 101 and the efg1ΔΔ/cph1ΔΔ mutant (Supplementary Fig. 1B). Yet, human and bovine albumin from different suppliers showed an overall tendency to significantly increase C. albicans-induced cytotoxicity (Supplementary Fig. 1C). Mouse albumin showed a strong tendency to increase cytotoxicity, while ovalbumin failed to enhance cytotoxicity (Supplementary Fig. 1C).

Using a luciferase cytotoxicity reporter cell line²³ we excluded that the albumin-enhanced cytotoxic potential of C. albicans strains was an artifact of the lactate dehydrogenase activity measurements (Supplementary Fig. 1D).

Collectively, these data suggest that albumin triggers an alternative pathogenicity pathway, independent of classical virulence mechanisms.

Increased C. albicans proliferation links to albumin-induced pathogenicity

Albumin was previously observed to increase C. albicans¹⁹ and C. glabrata¹⁷ growth. As fungal overgrowth often correlates with tissue damage during infection^{24, 25–26}, we investigated growth promoting potential in all strains tested. Similarly, all C. albicans clinical isolates and deletion mutants exhibited increased growth in the presence of albumin (Fig. 2A, B). Albumin fostered C. albicans 101 and efg1ΔΔ/cph1ΔΔ growth even without other nutrients being present (Supplementary Fig. 2A). Comparison of different multiplicities of infection (MOI) revealed that a MOI of 1 in the presence of albumin corresponded to the same level of cytotoxicity as a MOI of 100 without albumin for C. albicans 101 and efg1ΔΔ/cph1ΔΔ (Fig. 2C). Yet, separating the fungus from the epithelial layer, using transwells, showed that the albumin-augmented cytotoxic potential of 101 and efg1ΔΔ/cph1ΔΔ was contact-dependent, while albumin-augmented proliferation was not (Fig. 2D, Supplementary Fig. 2B). Interestingly, exposure of C. albicans to albumin just prior to infection was sufficient to boost pathogenicity (Fig. 2E). These findings suggest that the exposure to albumin, even short-term, reprograms C. albicans proliferation and cytotoxic potential.

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Fig. 2

Increased host cell cytotoxicity parallels fungal growth and is contact-dependent.

A Growth of different C. albicans isolates and deletion mutants in RPMI-1640 with or without albumin assessed by optical density measurements every 30 min for 45 h at 600 nm (n = 3). B Representative microscopic images showing SC5314, efg1ΔΔ/cph1ΔΔ, and 101 grown with and without albumin in the absence of host cells. Images were taken after 24 h at ×10 magnification (scale bar = 200 µm). C Cytotoxicity of A-431 cells infected with C. albicans strains at different multiplicities of infections (MOI) after 45 h post infection (hpi) with or without albumin (n = 3). D Comparison of cytotoxicity induced to A-431 cells in a transwell system (indirect infection) to directly infected A-431 cells in presence or absence of albumin after 45 hpi (n = 4). E Cytotoxicity of A-431 cells infected with albumin pre-incubated or non-albumin pre-incubated C. albicans strains. The pre-incubation took place for 0.5 h, 1 h, 2 h, or 3 h. The cytotoxicity measurements were carried out after 45 hpi, as a control served a direct infection with or without albumin (n = 4). Cytotoxicity (C–E) was quantified by measuring the LDH activity in the supernatant. Bars represent the mean ± SEM, and dots represent the mean of the technical replicates of the individual experiments. Data were tested for significance using a two-way ANOVA with Holm-Šídák multiple comparisons test (C–E). P values are provided in the figure for significant comparisons. Source data are provided as a Source Data file.

Albumin induces transcriptional reprogramming of C. albicans

To understand how albumin activates the pathogenicity and growth of C. albicans, transcriptional profiling was performed in the presence or absence of albumin during infection of A-431 cells. Principal component analysis (PCA) revealed that C. albicans reprograms its gene expression in the presence of albumin (Fig. 3A). Differential gene expression could be observed as early as 0.5 hpi, yet the reprogramed gene expression became more evident after prolonged infection (Fig. 3A). Overall, we observed more significantly downregulated genes in response to albumin at 0.5 hpi and 3 hpi, while the amount of significantly up- and downregulated genes was more balanced following prolonged infection (Supplementary Fig. 3A–C).

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Fig. 3

Albumin induces C. albicans transcriptional reprogramming.

C. albicans RNA sequencing was performed at 0.5 h, 3 h, and 24 h of infection of A-431 cells and albumin supplementation (5 mg/mL, n = 3 for each condition). A Principal component analysis of gene expression, with arrows indicating the impact of albumin (green), and infection duration (gray). B Significant Gene-Ontology (GO) term enrichment for differentially regulated genes (log₂fold change > 1 or < -1 and Benjamini-Hochberg P_adj < 0.01) at 3 h and 24 h. The numbers underneath the time points on X axis correspond to the counts of clusterProfiler (i.e., total number of genes assigned to GO categories). GeneRatio corresponds to the ratio between the number of input genes assigned to a given GO category and counts. Adjustment of P values is done by Benjamini-Hochberg procedure. C Expression of several virulence-associated genes. D Expression of genes involved in biofilm formation²⁷. Biofilm formation by C. albicans strains SC5314 and 101 on A-431 cells after 24 hpi was E quantified by measuring optical density at 550 nm after crystal violet staining (CV) staining (n = 3) or F visualized by confocal microscopy at ×10 magnification (scale bar =  50 µm). G Expression of amino acid and peptide transporters. Cytotoxicity of A-431 cells by measuring the LDH activity in the supernatant of (H) cells infected by C. albicans SC5314 or 101 in the presence or absence of amino acid supplementation (n = 4) or (I) infection by C. albicans SC5314, stp2ΔΔ, or stp2ΔΔSTP2 in the presence or absence of albumin (10 mg/mL) (n = 3). Legend color of C, D, G represent log₂ fold change of gene expression in presence vs. absence of albumin. Up- or downregulation is indicated by asterisks (Benjamini-Hochberg P_adj < 0.05 and log₂ fold change >1 or <–1). Bars represent the mean ± SEM, and dots represent the mean of the technical replicates of the individual experiments (E, H–I). E, H, I data were compared for significance using a two-way ANOVA with Holm-Šídák multiple comparisons test. P values are provided in the figure for significant comparisons. Source data are provided as a Source Data file.

To understand the pathways associated with the alternative pathogenicity induced by albumin, gene ontology (GO) enrichment analysis was performed (Fig. 3B and Supplementary Fig. 3D). At 3 hpi, up-regulated biological processes included translation, aerobic respiration, mitochondrial electron transport, as well as peptide transport (Fig. 3B). At 24 hpi, upregulated biological processes encompassed carbohydrate- and transmembrane transport, regulation of DNA, glycerol and arginine metabolic processes, regulation of filamentous growth, as well as biofilm formation and adhesion (Fig. 3B). Proteolysis and carbohydrate transport, as well as microautophagy of the nucleus were downregulated at 3 hpi and 24 hpi, whereas also GPI-anchor biosynthesis, DNA break repair, and aromatic amino acid catabolism were downregulated at 24 hpi (Fig. 3B).

Since the pathogenicity-inducing effect of albumin occurred independent of key virulence genes (Fig. 1E), we examined their expression. Interestingly, most of these genes were downregulated in presence of albumin (Fig. 3C, Supplementary data 1), further underscoring that albumin activates an alternative pathogenicity pathway. To understand the mechanisms of this alternative pathway, we focused on upregulated biological processes. We found many biofilm-associated genes²⁷ differentially regulated in response to albumin, especially genes associated with proliferation, biofilm maturation and production of extracellular matrix (ECM) (Fig. 3D, Supplementary data 1). In line with increased fungal proliferation (Fig. 2A, B), crystal violet staining and confocal microscopy confirmed that denser biofilms were formed by SC5314 and 101 in response to albumin (Fig. 3E, F).

In line with GO-term enrichment (Fig. 3B), we observed changes in expression of genes involved in amino acid and peptide transport (Fig. 3G, Supplementary data 1). This suggested that albumin is degraded into peptides^{28, 29–30} and amino acids that C. albicans tries to import. Nevertheless, exogenous supplementation of amino acids alone was not sufficient to mimic the alternative pathogenicity induced by albumin, instead it even reduced cytotoxicity (Fig. 3H). However, STP2, a transcription factor that regulates expression of amino acid permeases seemed to play an important role given that a stp2ΔΔ mutant showed attenuated cytotoxic potential (Fig. 3I). Contrastingly, deletion mutants of several other key genes differentially regulated in response to albumin, did not show an attenuated virulence phenotype in the presence of albumin (Supplementary Fig. 4A). Also, yeast- (SAP1-3) and hyphae-associated (SAP4-6) secreted aspartic proteases (SAPs) were dispensable for the alternative pathogenicity induced by albumin (Supplementary Fig. 4B). Moreover, both the fungus and epithelial cells can degrade albumin (Supplementary Fig. 4C). Suggesting that fungal protease activity is redundant for albumin-induced pathogenicity.

Overall, transcriptional responses of C. albicans to albumin suggest the induction of biofilm formation and metabolic changes to parallel the increased pathogenicity.

Albumin receptor and protein kinase signaling

Previously albumin was shown to bind C. albicans³¹, leading us to the idea that the fungus may use a specific receptor to induce the transcriptional changes and increase pathogenicity. We similarly observed binding of fluorescent albumin particularly to C. albicans hyphae, yet binding of albumin was almost absent in an als3ΔΔ mutant (Fig. 4A). However, since als3ΔΔ also can be augmented in its cytotoxic potential by albumin (Fig. 1E), we believe Als3 may merely serve as a scavenging receptor.

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Fig. 4

Albumin-induced cytotoxicity relies on fungal iron acquisition.

A Binding of albumin (Alexa Fluor 647 conjugated; 0.1 mg/mL) after 6 h at 37 °C and 5% CO₂ to C. albicans WT (BWP17/Clp30) and an als3ΔΔ mutant. Images were taken after 6 h at ×64 magnification (scale bar = 20 µm). Representative images of n = 3 experiments. B Differentially expressed protein kinase genes. C Growth of C. albicans protein kinase deletion mutants and WT strain in RPMI-1640 with or without albumin assessed by optical density measurements every 30 min for 24 h at 600 nm (n = 3). Cytotoxicity of A-431 cells infected with C. albicans protein kinase deletion mutants and WT strains with or without albumin (D–F) and/or 50 μM BPS (G) after 45 h post infection (hpi) (D, F, Gn = 3, E uninfected and SC5314 n = 12, ire1ΔΔ, sln1ΔΔ, sch9ΔΔ, rim1ΔΔ, cek2ΔΔ, and ksp1ΔΔ n = 6). Cytotoxicity was quantified by measuring the LDH activity in the supernatant. Bars represent the mean ± SEM, and dots represent the mean of the technical replicates of the individual experiments (D–G). E–G Data were compared using a two-way ANOVA with Holm-Šídák multiple comparisons test. P values are provided in the figure for significant comparisons Source data are provided as a Source Data file.

Virtually all signaling pathways processing external signals and driving cellular changes in C. albicans involve protein kinases³². Interestingly, around 20% of all predicted protein kinase genes in C. albicans showed a strong differential expression in response to albumin, suggesting their potential involvement in the cellular changes by albumin (Fig. 4B, Supplementary data 1). We screened deletion mutants of the differentially expressed protein kinases for their ability to change their growth in response to albumin (Fig. 4C). We identified deletion mutants that were unable to damage A-431 cells (Fig. 4D), of which growth was restored (sln1ΔΔ) or not restored (ire1ΔΔ) by albumin. We selected several additional mutants (rim11ΔΔ, ksp1ΔΔ, and cek2ΔΔ) for further validation (Fig. 4D), yet all mutants, except ire1ΔΔ, were boosted by albumin (Fig. 4E). Ire1 has a conserved role in the unfolded protein response by cleaving HAC1 mRNA in response to endoplasmic reticulum stress³³. Cytotoxicity of hac1ΔΔ could still be increased with albumin to some extent (Fig. 4F), which suggests that Ire1 partially acts in a Hac1-dependent manner in the adaptation to albumin. Ire1 was shown to be indispensable for C. albicans growth under iron-limiting conditions independently of Hac1³⁴. Together with the downregulation of iron transport genes observed at 3 hpi (Fig. 3B) this suggests a role for iron in the albumin-augmented cytotoxicity. In line with this, iron chelation reduced the capacity of albumin to enhance cytotoxic potential of SC5314 (Fig. 4G). This suggests that albumin induces iron metabolism in C. albicans via Ire1.

Albumin re-wires C. albicans metabolism

To follow up on the differential expression of metabolic pathways (Fig. 3B), intracellular and extracellular metabolic profiling was performed of C. albicans grown in absence of host cells. Exposure to albumin changed intracellular metabolite pools as early as 3 h after exposure, which became more pronounced after 24 h (Fig. 5A, Supplementary Fig. 5A, Supplementary data 2). The exometabolome also showed significant changes depending on the presence of albumin (Fig. 5B, Supplementary Fig. 5A, Supplementary data 3). Unsupervised hierarchical clustering revealed groups of metabolites differentially impacted by albumin. Intracellular metabolites that were depleted upon albumin treatment (cluster 1 and cluster 4) consisted predominantly of carbohydrates and lipids, respectively (Fig. 5C, D; Supplementary data 2). Whereas intracellular metabolites in cluster 5 and 6 rather tended to accumulate upon albumin treatment and consisted predominantly of energy metabolites and co-factors (cluster 5) or lipids (cluster 6) (Fig. 5D). In the exometabolome, a specific group of metabolites were consumed from culture medium exclusively when albumin was there (cluster 4) (Fig. 5E), which ranged from amino acids and nucleotides to energy metabolites and co-factors (Fig. 5E, F, Supplementary data 3). In contrast, the presence of albumin was associated with an enrichment of particularly lipids and some specific amino acid metabolites extracellularly (cluster 3; Fig. 5E, F). Overall, albumin significantly changed levels of 151 and 138 intracellular metabolites at 3 h and 24 h, respectively, and 49 exometabolites at 24 h (Fig. 5G, and Supplementary Fig. 5A). Notably, lipids were the largest class of metabolites found in the exometabolome (Fig. 5G).

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Fig. 5

Albumin changes C. albicans intracellular and extracellular metabolite pools.

Principal component analysis (PCA) of A the intracellular metabolite composition in C. albicans SC5314 after 3 h, and 24 h, and B the exometabolome after 24 h growth of C. albicans with and without albumin in RPMI-1640 (5 mg/mL; n = 5 for each time point). Arrows indicate the albumin (green) or time (gray) dependent effect on the metabolic profile. C Unsupervised hierarchical clustering based on the Euclidean distance of relative metabolite abundance of the intracellular metabolome after 3 h and 24 h. D Metabolite composition of the clusters altered by the presence of albumin. E Unsupervised hierarchical clustering based on the Euclidean distance of relative metabolite abundance of the exometabolome after 24 h. F Metabolite composition of the clusters altered in response to albumin presence. G Differentially (P < 0.05 and log₂ fold change >1 or <-1) accumulated or depleted metabolites in response to albumin found throughout the different conditions. Source data are provided as a Source Data file.

To understand how the cellular metabolic network in C. albicans was rewired by albumin, we overlaid the changes of the intracellular metabolites with the KEGG metabolic pathways map 01100 (Fig. 6A, Supplementary Fig. 6)³⁵. This revealed that albumin impacted a variety of pathways and led to an extracellular accumulation of different fatty acids. In line with the enriched biological processes on a transcriptional level (Fig. 3B), glycolysis metabolites were significantly reduced and TCA-cycle metabolites and amino acids were significantly increased (Fig. 6B). Associated with this shift in TCA activity, higher abundance of energy-carriers NADH, NADPH, and acetyl-CoA suggested a more-efficient metabolism upon exposure to albumin (Fig. 6C). In line with this, we measured increased respiration of C. albicans following exposure to albumin (Fig. 6D). Interestingly, metabolites of the glutathione pathway, an important pathway for detoxification of reactive oxygen species, were increased in abundance when C. albicans was grown in the presence of albumin (Fig. 6B). Palmityl-CoA, coming from fatty acid oxidation and biosynthesis, accumulated in response to albumin (Fig. 6C). Furthermore, metabolites of the phosphatidylcholine metabolism were more abundant intracellularly (Fig. 6B). Contrastingly, poly- and mono-unsaturated fatty acids such as arachidonic acid, linolenic acid, or alpha-linolenic acid decreased in their intracellular abundance when albumin was present (Fig. 6B). Interestingly, many of the metabolites belonging to unsaturated fatty acids were among the top increased metabolites in the exometabolome (Figs. 6A, E, Supplementary Fig. 5B).

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Fig. 6

Albumin rewires metabolic pathways in C. albicans.

A Differential changes in intracellular metabolite abundance plotted on the KEGG metabolic pathways map (01100)³⁵ using iPath3¹¹². Differential changes upon exposure to albumin at 24 h are shown in the brown-petrol scale with pathway width highlighting the significance. Metabolites significantly enriched or depleted in the exometabolome are highlighted using the red-blue color scale all with the same width. Relevant intracellular pathways are annotated in orange boxes, intracellular energy carrying metabolites in blue boxes, and extracellular fatty acid metabolites with a green box. Data were tested for significance using a two-tailed Welch’s t-test. B Normalized abundance (measured abundance normalized to median of metabolite) of all detectable metabolites belonging to individual intracellular metabolic pathways in the presence or absence of albumin after 3 h and 24 h (data combined from all metabolites of the corresponding pathway glycolysis = 9, TCA-cycle = 9, amino acids = 20, PPP = 11, Phosphatidylcholines = 21, glutathione  = 13, MC/LC fatty acids = 16 from n = 5 replicates). C Normalized abundance of metabolites involved in energy metabolism after 3 h and 24 h (n = 5). D Cellular respiration of C. albicans (SC5314 and 101) after 4 h of pre-incubation in presence or absence of albumin (10 mg/mL; n = 3). E Normalized abundance of all detectable metabolites from fatty acid pathways of the exometabolome after 24 h (data combined from all metabolites of the respective pathway medium chain fatty acids = 4, polyunsaturated fatty acids = 6, unsaturated fatty acids = 2 from n = 5 replicates). Bars in (D) represent the mean ± SEM, dots represent the mean of the technical replicates of the individual experiments. Lines in (B, C, E) represent the mean and dots represent measured metabolites in the individual experiments. Data were tested for significance using two-way ANOVA with Holm-Šídák multiple comparisons tests. P values are provided in the figure for significant comparisons. Source data are provided as a Source Data file.

C. albicans secreted lipids cause cytotoxicity

We hypothesized that the reprogramming of C. albicans metabolism may underlie the increased cytotoxicity. Rapid glucose consumption was previously identified as a mechanism triggering macrophage lysis³⁶. Nevertheless, even in the absence of glucose, albumin increased pathogenicity of 101, and an efg1ΔΔ/cph1ΔΔ deletion mutant (Supplementary Fig. 7A). Apart from consuming essential nutrients, extracellularly accumulating metabolites, may cause cytotoxicity. With exception of p-cresol sulfate (a known protein degradation product in bacterial metabolism³⁷) and 4-hydroxphenylpyruvate, the top accumulating metabolites belonged to mono- and polyunsaturated fatty acids (Fig. 6E, Supplementary Fig. 5B, Supplementary data 3). These metabolites were introduced to our in vitro infection model, to analyze their cytotoxic potential in isolation. During infection of A-431 cells, only the introduction of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) increased C. albicans-induced cytotoxicity (at 20 µM) to a similar extent as albumin (Fig. 7A, B, Supplementary Fig. 7B). This connects the oxylipin 13-HODE with the enhanced cytotoxicity induced by albumin.

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Fig. 7

Albumin-enhanced cytotoxicity is linked with oxylipin 13-HODE.

A, B Cytotoxicity of A-431 cells induced by secreted mono- or polyunsaturated fatty acids and albumin, with and without C. albicans (101) infection after 45 h (n = 3). C Detection of mono- and polyunsaturated fatty acids and oxylipins of culture medium with or without albumin (n = 3). D Detection of 13-HODE during A-431 cell infection with C. albicans (SC5314) in the presence or absence of albumin (n = 3) E Cytotoxicity of A-431 cells infected with C. albicans (SC5314, efg1ΔΔ/cph1ΔΔ, and 101) with and without FAF albumin at 45 hpi (n = 3) (first panel). Detection of 13-HODE (second panel), AA (third panel), and DHA (fourth panel) production by C. albicans (SC5314 and 101) in the presence or absence of FAF albumin (n = 3). F Detection of 13-HODE production by C. albicans (101) in the presence of indicated serum proteins (n = 3) in absence of A-431 cells. G Detection of 13-HODE production by C. albicans (efg1ΔΔ/cph1ΔΔ) with and without albumin (n = 3) in absence of A-431 cells. H Detection of 13-HODE production by C. albicans deletion mutants and revertant strains, with albumin (n = 3) during infection of A-431 cells. Cytotoxicity (A, B, E) was quantified by measuring the LDH activity in the supernatant. Bars represent the mean ± SEM, and dots represent the mean of the technical replicates of the individual experiments (B–H). Data were tested for significance using two-way ANOVA with Holm-Šídák multiple comparisons tests (B–E), one-way ANOVA with Holm-Šídák multiple comparisons tests (F, H) or paired, two-tailed parametric t test (G). P values are provided in the figure for significant comparisons. AA Arachidonic acid, DHA Docosahexaenoic acid, EPA Eicosapentaenoic acid, LA Linoleic acid, 9- or 13-HODE 9- or 13-hydroxy-9Z,11E-octadecadienoic acid, FAF albumin: fatty acid free albumin. Source data are provided as a Source Data file.

As albumin can bind and transport fatty acids³⁸, we wanted to ensure that accumulating 13-HODE is fungal-derived and not introduced by albumin supplementation. In the absence of both C. albicans and host cells, targeted metabololipidomics on albumin-supplemented medium revealed the accumulation of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), but not the oxylipin 13-HODE (Fig. 7C). This suggests that C. albicans produced 13-HODE specifically upon albumin treatment (Fig. 7D). Furthermore, 9/13-HODE and precursor fatty acids are found intracellular in C. albicans regardless of albumin being present (Supplementary Fig. 5C).

To exclude the possibility that albumin-increased cytotoxicity is merely due to albumin-bound fatty acids fueling fungal lipid metabolism, we supplemented fatty acid-free albumin. This augmented C. albicans pathogenic potential and facilitated 13-HODE production similar to normal albumin (Fig. 7E). Contrastingly, other serum proteins that, at physiological albumin concentration (10 mg/mL), did not enhance C. albicans cytotoxic potential (Supplementary Fig. 1B) also failed to stimulate 13-HODE production in C. albicans (Fig. 7F). SC5314 (Fig. 7D), 101 (Fig. 7E) as well as the yeast locked efg1ΔΔ/cph1ΔΔ mutant (Fig. 7G) increased 13-HODE production in response to albumin. The C. albicans deletion mutants, in which albumin did not strongly augment cytotoxicity (stp2ΔΔ; Fig. 3I) and (hac1ΔΔ; Fig. 4G) or failed to augmented cytotoxicity (ire1ΔΔ; Fig. 4G), also showed significantly diminished or abolished 13-HODE production, respectively (Fig. 7H).

In human cells, linoleic acid (LA) can be oxidized by lipoxygenases (LOXs) to 9- or 13-HpODE³⁹. Reduction of these intermediate hydroperoxides by glutathione peroxidase forms 9- and 13-HODE, respectively³⁹. In C. albicans, the pathway of 13-HODE biosynthesis has not been resolved. Our metabolome data suggested increased activity of the glutathione pathway in the presence of albumin (Fig. 6B), which may facilitate the last step in 13-HODE formation. While 15-LOX is not known to be expressed in C. albicans, nordihydroguaiaretic acid (NDGA) that inhibits the catalytic cycle of human 15-LOX, also effectively decreased 13-HODE release by C. albicans (Fig. 8A), as well as albumin-induced C. albicans cytotoxicity (Fig. 8B) without diminishing fungal growth (Fig. 8C). Supplementation of a different antioxidant, N-acetyl cysteine (NAC), also significantly reduced albumin-induced C. albicans cytotoxicity, yet not to the magnitude of NDGA (Fig. 8D). Contrastingly, inhibitors for cyclooxygenase (Acetylsalicylic acid, ASA) and phospholipase A2 (varespladib), which are involved in pathways leading to other oxylipins, did not impair albumin-induced C. albicans cytotoxicity (Fig. 8E). Previously, 13-HODE has been shown to modulate cell death through signaling via peroxisome proliferator-activated receptor (PPAR)γ⁴⁰. Inhibition of PPARγ led to a mild reduction of C. albicans cytotoxicity (Fig. 8F). This may suggest some involvement of PPARγ-mediated signaling, though other PPARγ-independent effects of 13-HODE also likely play a role^41,42.

[See PDF for image]

Fig. 8

Inhibition of 13-HODE biosynthetic and signaling pathways results in reduced epithelial cell cytotoxicity.

A Detection of 13-HODE production during A-431 cells infection with C. albicans (101), with and without albumin, in the presence of a known inhibitor of 15-LOX in humans (NDGA, 100 μM) (n = 4). B Cytotoxicity of A-431 cells infected with C. albicans (101), with and without albumin, in the presence of NDGA (100 μM) at 45 h post infection (hpi) (n = 7). C Fungal colony forming units (CFU) per mL of the C. albicans 101, with and without albumin (10 mg/mL), in the presence of NDGA (100 μM) (n = 3). D Cytotoxicity of A-431 cells infected with C. albicans (101), with and without albumin, in the presence of the antioxidant NAC (250 μM) at 45 hpi (n = 5). E Cytotoxicity of A-431 cells infected with C. albicans (101), with and without albumin, in the presence of known inhibitors of the enzyme cyclooxygenase (ASA, 100 μM) and phospholipase A2 (Varespladib, 100 μM) at 45 hpi (n = 3). F Cytotoxicity of A-431 cells infected with C. albicans (101), with and without albumin, in the presence of a known PPARγ inhibitor (GW9662, 10 μM) at 45 hpi (n = 4). Cytotoxicity (B, D–F) was quantified by measuring the LDH activity in the supernatant. Bars represent the mean ± SEM, and dots represent the mean of the technical replicates of the individual experiments. Data were tested for significance using two-way ANOVA with Holm-Šídák multiple comparisons tests (A, B, D–F). P values are provided in the figure for significant comparisons. NDGA Nordihydroguaiaretic acid, NAC N-acetyl cysteine, ASA Acetylsalicylic acid, PPARγ Peroxisome proliferator activated receptor gamma. Source data are provided as a Source Data file.

Discussion

We demonstrated that the pathogenicity of clinical C. albicans isolates, and mutants lacking essential virulence genes can be restored in the presence of the abundant host protein albumin in in vitro infection models. This effect was associated with enhanced fungal growth, and metabolic and transcriptional changes in C. albicans. The presence of albumin led to the release of oxylipins by C. albicans, particularly 13-HODE, which we linked to increased epithelial cytotoxicity. Our data underscore that in the presence of albumin, C. albicans can exert pathogenicity through mechanisms independent of known virulence factors.

Our findings challenge the view that filamentation is essential for C. albicans virulence potential⁷. While the importance of filamentation and candidalysin has been confirmed in a wide variety of infection models^{3,7,43, 44–45}, the low ex vivo pathogenincity of clinical isolates from infections contradicts this dogma. We observed that albumin enhanced pathogenicity across different strains, including previously considered avirulent mutants⁷. This implies that host factors play a pivotal role in virulence potential in vivo. Moreover, an eed1ΔΔ deletion mutant was considered non-pathogenic based on in vitro infection models^10,46. Yet, during intravenous infection of mice, eed1ΔΔ exhibited a high virulence potential¹⁰. This was associated with rapid proliferation due to metabolic adaptation and improved fitness, resulting in high fungal burden in vital organs¹⁰. Another study showed that a slf2ΔΔ mutant, incapable of filamentation, was avirulent during epithelial infection but not during systemic infection⁴⁷. The fungal morphology observed in the infected kidneys exhibited the yeast phenotype⁴⁷. This suggests an alternative pathogenicity mechanism to the formation of long invasive hyphae and candidalysin secretion. The intravenous infection route exposes fungal cells to high concentrations of albumin and other serum proteins. Our insights into how albumin induces metabolic reprogramming and augments fungal proliferation can contribute to explain the restored virulence of C. albicans eed1ΔΔ in vivo. We showed that the yeast-locked mutant strains efg1ΔΔ/cph1ΔΔ and eed1ΔΔ can cause host cytotoxicity in vitro when albumin is present. Yet, in contrast to eed1ΔΔ, efg1ΔΔ/cph1ΔΔ mutants are avirulent during systemic and catheter infection models, suggesting that some degree of morphology changes as in eed1ΔΔ, or genes under control of efg1ΔΔ/cph1ΔΔ are required in vivo^7,48. In line with this, EFG1 was the only examined virulence gene that rather showed a tendency towards upregulation in our transcriptional data set. The avirulence of efg1ΔΔ/cph1ΔΔ in vivo does not necessarily have to be associated with pathogenicity towards host tissues but may rather relate to a reduced fitness during encounters with immune cells. The fact that albumin is a driver of pathogenicity in vivo still requires confirmation. To this end albumin-deficient mice may be able to provide important insights⁴⁹.

We observed that albumin also restored the cytotoxicity of deletion mutants exhibiting normal hyphae formation but lacking hyphae-associated virulence genes (als3ΔΔ, ece1ΔΔ). This underscores that the albumin-induced cytotoxic potential is independent of hyphal or yeast morphology and relies on pathways expressed in both morphotypes. C. albicans induces a third morphology, known as pseudohyphae, which generally are less virulent compared to true hyphae⁵⁰. We observed that clinical strains forming pseudohyphae benefit from albumin, displaying cytotoxicity levels similar to those of the filamentous reference strain SC5314. Hereby, our study contributes to the discussion that the ability to morphologically switch between yeast and hyphal forms, rather than relying on just one form, is crucial for pathogenicity^10,51,52.

Albumin induced drastic transcriptional and metabolic changes in C. albicans. The cellular metabolism seemed to deviate from glycolysis to lipid and amino acid utilization, increased TCA-cycle activity, and respiration. Crucial metabolites for energy production accumulated, suggesting more metabolically active fungal cells⁵³, which was linked to enhanced fungal growth and biofilm formation. Yet, the downregulation of key adhesins HWP1 and ALS3 could suggest that albumin-induced biofilms are less stable. The presence of albumin induced the upregulation of numerous amino acid and peptide transporters implying that C. albicans tries to utilize albumin in smaller units. C. glabrata relies on albumin proteolysis by A-431 cells to activate its pathogenicity¹⁷. Macropinocytosis facilitates albumin uptake into epithelial cells, where the protein is degraded and subsequently secreted^17,54. While C. albicans may also benefit from this mechanism, it possesses a family of secreted aspartyl proteinases capable of degrading albumin^{28, 29–30}. Yet, mutants deficient in SAP1-3, SAP4-6 or SAP9-10 were not compromised in their response to albumin, suggesting redundancy between fungal yeast and hyphae associated proteases as well as host proteases. Our metabolome data suggest that the increased amino acid and peptide availability may fuel the fungal energy metabolism including the TCA-cycle. In line with this, several amino acids have been shown to drive TCA-cycle activity^55,56. The ability to adapt to different local nutrients by C. albicans and its metabolic plasticity, is not only connected to enhanced fungal growth, but also involves enhanced stress resistance, cell wall remodeling, or other key virulence factors^10,53,57. We propose metabolic reprogramming-induced enhanced pathogenicity.

As potential executing metabolites of the enhanced cytotoxic potential we identified oxidized polyunsaturated fatty acids, such as 13-HODE, in supernatants of albumin-treated C. albicans. These oxidized fatty acids are also called oxylipins, and can be produced by vertebrates, fungi, or plants⁵⁸. Due to their roles as effector molecules these are often termed as bioactive lipid mediators. It has been suggested that oxylipins produced by the host and microbes could interfere in inter-species metabolism, perception or signaling processes⁵⁸. We found that 13-HODE was specifically produced by C. albicans in the presence of albumin, even fatty acid free albumin. It caused cytotoxicity at concentrations where other accumulating metabolites did not. Interestingly, extracellular 13-HODE was reported to induce mitochondrial dysfunction and airway epithelial apoptosis, which was associated with its interaction with the transient receptor potential cation channel, vanilloid type 1 (TRPV1) receptor⁵⁹. In colorectal cancer cells, 13-HODE was shown to signal through PPARγ, inducing colorectal cell apoptosis⁴⁰. Blockade of PPARγ moderately, yet significantly reduced cell death induced by C. albicans in the presence of albumin. The inability to fully restore epithelial cytotoxicity is likely due to involvement of other pathways and having cross-talk with 13-HODE signaling⁶⁰. 13-HODE has also been shown to directly inhibit catalase⁴¹, which may compromise the capacity of host cells to detoxify ROS. Furthermore, 13-HODE was also found to induce ROS and ER-stress in hepatocytes⁴². This was found to be rescued by supplementation of N-acetyl cysteine, which also slightly reduced the albumin-augmented C. albicans cytotoxicity. Increased fungal growth and biofilm formation coupled with 13-HODE accumulation seemed to drive the epithelial cytotoxicity during C. albicans infection in the presence of albumin. The biofilm formation can form a microenvironment that is favorable for the concentrated accumulation of 13-HODE in proximity to host cells. This could explain the observed lack of cytotoxicity upon physical separation of the fungus and A-431 cells. Further, the cytotoxic potential of exogenously added 13-HODE was primarily observed when the fungus was also present. This suggests that 13-HODE may synergize with actions from the fungus for its cytotoxic potential. These could be mechanisms like cell starvation, candidalysin (in strains capable of producing it), and fungal proteases.

In humans, the biosynthesis of 13-HODE requires LOX enzymes, glutathione peroxidases^{39,61, 62–63}, or non-enzymatic processes^39,64. To our knowledge, no LOX enzymes have been identified in C. albicans, along with the inability to detect a homologous sequence in its genome^65,66. Several studies have shown the production of oxylipins by C. albicans, such as prostaglandins^{65, 66–67}, which can be influenced by LOX-inhibitors^65,68,69. NDGA, which exerts its inhibitory effects through interfering with the catalytic iron of LOX enzymes⁷⁰, could reduce the levels of released 13-HODE and abolish the cytotoxicity-boosting effect of albumin. Moreover, Ire1-mediated processes such as fungal iron metabolism seem also crucial for the albumin-enhanced pathogenicity. Therefore, we hypothesize that enzymes analogous to 15-LOX with corresponding sensitivity to NDGA are present in C. albicans. As glutathione transferases and peroxidases are involved in 13-HODE synthesis^39,63, the significant increase of glutathione metabolism may be linked with 13-HODE production during albumin supplementation. It is not uncommon that oxylipin biosynthesis pathways differ between humans and C. albicans. Exemplary, the fungus can synthesize PGE₂ using a fatty acid desaturase (Ole2) and multicopper oxidases (Fet3 and Fet31)^65,71, whereas in humans this relies on cyclooxygenases⁷².

It remains unresolved how albumin exactly drives 13-HODE production in C. albicans. Linolenic acid bound to albumin could serve as an intermediate substrate. In line with this, the bacterium Peptostreptococcus magnus was shown to  scavenge fatty acids from albumin, and enhances its growth and virulence^73,74. Similarly, C. albicans has been suggested to exploit arachidonic acid from the host⁷⁵. However, since fatty acid-free albumin also potentiated cytotoxicity and lead to release of different lipids including 13-HODE, it may be that C. albicans synthesizes the substrates for oxylipin production itself.

To our knowledge, the 13-HODE production was not yet described for C. albicans. However, the opportunistic pathogens Aspergillus fumigatus^76,77 and Pseudomonas aeruginosa⁷⁸ were found to produce 13-HODE. In the context of airway epithelial injury, 13-HODE was shown to induce hyperresponsiveness, cell damage, and neutrophilia^59,79. During infection, granulocytes also can contribute to 13-HODE production⁸⁰, especially eosinophils when challenged with pathogenic fungi⁸¹. It also has been shown that 13-HODE can influence the chemotaxis of neutrophils⁸². As increased hyperresponsiveness and neutrophilia are driving forces of immunopathology associated with Candida infections^{10,83, 84–85}, it is tempting to speculate that albumin-induced 13-HODE release by the fungus may be a contributing factor.

By supplementing albumin, we address the essential need to design infection models more physiologically relevant and improve their translational capacity to human disease. Previously, we observed that albumin can neutralize candidalysin and with this cytotoxicity of the strain SC5314 for which the induction of cell damage strongly relies on this toxin¹⁹. Here, we observed that during early infection this extends to other strains exhibiting strong cytotoxicity on HEK293A cells. However, for C. albicans clinical isolates that were considered avirulent ex vivo, the introduction of albumin reconstituted the pathogenic phenotype aligning with the clinical origins of the strains. This underscores that mimicking a more physiological environment enables a more accurate representation of pathogenic potential in in vitro infection models. Similarly, sensitivity to antifungal treatment may be more accurate when using more physiological models⁸⁶. A plethora of physiological modifications, such as physiological human body temperature, CO₂ levels, and pH values⁸⁷, are indispensable for cell culture maintenance. However, further modifications such as the inclusion of serum or -proteins^{11,17,19,88, 89–90}, adjusting physiologically relevant carbon sources and their concentrations^{91, 92–93}, or the co-cultivation with common commensals to mimic the host-environment^{94, 95, 96, 97–98} can help to further improve translational capacity. Many factors have already individually been shown to impact C. albicans pathogenicity, or the interaction with the immune system^11,19,91,99. For example, we previously found that the serum protein α1-antitrypsin can increase C. albicans pathogenic potential through induction of filamentation¹¹.The rapid development of organoid and organ-on-chip models poses a promising avenue to make leaps forward in a more physiological modeling of human disease using in vitro models¹⁰⁰.

Here, we present a comprehensive perspective of how albumin-induced transcriptional and metabolic changes in C. albicans drive an alternative pathway of fungal pathogenicity. This was linked to proliferation, biofilm formation, and the accumulation of the oxylipin 13-HODE. These findings highlight the crucial role of host proteins in fungal pathogenicity.

Methods

C. albicans strains and culture conditions

All C. albicans strains used in this study are presented and described in Supplementary Table 1. To inoculate overnight cultures, single colonies were picked from yeast peptone dextrose (YPD) agar plates and grown in 5 mL liquid YPD medium using a 25 mL glass Erlenmeyer flask. Cultures were incubated overnight in an orbital shaker at 180 rpm and 30 °C. Yeast cells were harvested by centrifugation (20,000 × g, 1 min), washed three times with phosphate-buffer saline (PBS) pH 7.4 and adjusted to 4 × 10⁷–2 × 10⁷ yeast cells per milliliter (cells/mL) in PBS (pH 7.4). The final dilution and the respective culture medium varied depending on the carried-out assay.

For the protein kinase deletion mutant screen, strains were grown in 1 mL liquid YPD medium using a 24-well plate and incubated for overnight at 30 °C with shaking at 180 rpm. Plates were then centrifuged at 4000 × g and washed twice with PBS (pH 7.4).

Chemicals and proteins

All albumin preparations used in this study are listed in Supplementary Table 2. Other compounds are listed in Supplementary Table 3. 13-HODE is delivered pre-dissolved in ethanol, which can lead to cell toxicity. Therefore, the ethanol was evaporated and 13-HODE was dissolved in DMSO, which proofed to be non-toxic in the used concentrations compared to ethanol. For all inhibitors dissolved in DMSO, control infections with an equal amount of DMSO were performed to exclude cytotoxicity induced by DMSO alone.

Live-cell imaging

To identify the morphology of the clinical strains, a 96-well plate was incubated with 2 × 10⁴ yeast cells per well (cells/well) at 37 °C with 5% CO₂ in RPMI-1640 the presence or absence of 10 mg/mL albumin. Images were taken at ×10 or ×40 magnification after 24 h with the Axio Observer microscope (Carl Zeiss Microscopy) and exported with Zeiss Zen3.1 (blue edition).

Mammalian cell cultivation

All cell lines were handled according to the supplier instructions. In brief, a human vulvar epithelial cell line A-431 (Deutsche Sammlung von Mikroorganismen und Zellkulturen; DSMZ no. ACC 91) was used as the main in vitro infection model. A-431 cells were maintained in RPMI-1640 medium with L-glutamine (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bio & Sell) at 37 °C and 5% CO₂. For experiments, A-431 cells were seeded in 96-well plates (TPP^TM) at 2 × 10⁴ cells per well (cells/well), in 24-well plates at 1 × 10⁵ cells/well, and in 6-well plates at 3 × 10⁵ cells/well. A-431 cells were cultured for 2 days at 37 °C and 5% CO₂ prior to infection. Kidney epithelial cell line HEK293 subclone HEK293A (human embryonic kidney cells transformed with sheared human adenovirus type 5 DNA) were obtained from Invitrogen. The cells were maintained in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS at 37 °C and 5% CO₂.

The cell lines have been authenticated via commercial STR profiling (Eurofins Genomic) and regularly checked for mycoplasma contaminations using a PCR mycoplasma test kit (PromoKine) according to the protocol supplied by the manufacturer.

In vitro epithelial infection model

For our infections, the medium was removed and replaced with prewarmed fresh RPMI-1640 (or RPMI-1640 with L-glutamine but without glucose, catalog number 11879020, Thermo Fisher Scientific) without FBS, and supplemented with or without 10 mg/mL albumin. A-431 cells were subsequently infected with the desired C. albicans strain at a multiplicity of infection (MOI) of 1. This resembles 2 × 10⁴ yeast cells per well in 96-well plates and 8-well µ-slides or 1 × 10⁵ yeast cells per well in 24-well plates. For the infection of 6-well plates, a total concentration of 3 × 10⁶ or 6 × 10⁷ yeast cells/well, depending on the time point, was added (see RNA isolation). For transwell assays, A-431 cells seeded in 24-well plates were covered with 750 µL fresh RPMI-1640 medium with or without 10 mg/mL albumin. Transwell inserts (polycarbonate membrane inserts with 0.4 µm pore size; Corning) were placed in the well and filled with 250 µL of the desired C. albicans strain (1 × 10⁵ yeast cells/transwell) in RPMI-1640 medium with or without 10 mg/mL albumin.

For pre-incubation experiments, 2 mL of 1 × PBS (pH 7.4) containing a total of 8 × 10⁵ yeast cells were incubated with or without 10 mg/mL albumin for 0.5 h, 1 h, 2 h, and 3 h. Afterwards, the cells were centrifuged (20,000 × g, 10 min), washed with 1 × PBS, re-counted, and adjusted to 4 × 10⁵ yeast cells/mL in the respective medium. The pre-incubated yeast cells were used for A-431 cells infection in a 96-well plate as described above. As a control A-431 cells were directly infected in the presence or absence of 10 mg/mL albumin (0 h control).

If not stated otherwise, cell lines were infected for 18 h or 45 h at 37 °C and 5% CO₂.

Cytotoxicity assay

Upon necrotic cell damage cells lose membrane integrity and cytoplasmic contents can leak out. As a marker for host cell cytotoxicity, the activity of the ubiquitous cytoplasmic enzyme lactate dehydrogenase (LDH) was quantified in supernatants of infected A-431 cells after 18 and 45 hpi using a Cytotoxicity Detection Kit (Roche) according to the manufacturer’s protocol. The measurements were performed as duplicates or triplicates, and values were plotted as LDH release in ng/mL.

Luciferase cytotoxicity reporter cell line

As another cytotoxicity readout, a luciferase reporter cell line was used²³. In brief, infected A-431-Nluc plates were centrifuged at 250 × g for 10 min. Then 100 µL of supernatant was collected in a fresh plate. For the luciferase assays, 5 µL of the supernatant was diluted with 95 µL PBS (pH 7.4) in white flat-opaque-bottomed 96-well plates (Thermo Fisher Scientific). Subsequently, 100 µL of coelenterazine (10 µM in PBS pH 7.4) was added, and the luciferase activity was determined using a luminescence microplate reader (Tecan Infinite M-Plex; i-control software), with integration time of 100 milliseconds and OD2 compensation. The measurements were performed as duplicates or triplicates.

Microscopic analysis of bound and degraded albumin

To detect the binding capacity of albumin to C. albicans, 8-well µ-slides were incubated with 1 × 10⁴ yeast cells/well and incubated with 0.1 mg/mL BSA Alexa Fluor^TM 647 (Jackson ImmunoResearch) at 37 °C and 5% CO₂. Bright field and fluorescent (650/668 nm) images were taken at ×64 magnification after 6 h with an Axio Observer microscope (Carl Zeiss Microscopy). To detect the degradation process of albumin, 8-well µ-slides containing wells with and without A-431 cells were infected with 2 × 10⁴ yeast cells/well supplemented with 0.1 mg/mL DQ^TM Red BSA (labeled with BODIPY® TR-X dye; Invitrogen). After 24 hpi, each well was washed, fixed with 4% Histofix (Roth), mounted with ProLong Gold antifade mountant (Thermo Fisher Scientific), and visualized at ×64 magnification with an Axio Observer microscope (Carl Zeiss Microscopy) using bright field and fluorescence channels (596/621 nm).

C. albicans growth quantification

Fungal growth was quantified in selected infection experiments using a previously described CFU plating method¹⁷. In brief, well contents were scraped, washed twice with 1 mL PBS to ensure the detachment of all host and fungal cells. After every step, the content was transferred into a collection tube. For transwell experiments, inserts were placed in 50 mL centrifuge tubes containing 10 mL 1 × PBS (pH 7.4), and vortexed to separate the fungal cells from the membrane. To identify the growth inducing capacity of albumin as sole nutrient source, C. albicans yeast cells (4 × 10⁵ yeast cells/mL) were pre-incubated with or without 10 mg/mL albumin in 1 mL PBS for 0 h, 3 h, 6 h, or 18 h at 30 °C, and directly used for the CFU assay. For all collected fungal cell suspensions serial dilutions (1:100, 1:1000, or 1:10,000) were plated on YPD agar plates. Single colonies were counted after two days of incubation at 30 °C. Values are represented as CFU/mL of the original collection tube. Fungal growth in the absence of host cells was determined via optical density (OD) at 600 nm. C. albicans strains (1 × 10⁴ yeast cells/well) were incubated with and without 10 mg/mL of albumin at 37 °C with 5% CO₂ in a microplate reader (Tecan Infinite M-Plex; i-control software). Growth was monitored with OD₆₀₀ measurement every 30 min over 45 h.

Biofilm formation

Biofilm formation in the presence or absence of albumin (10 mg/mL) was quantified using a crystal violet (CV) assay based on a previously described method¹⁰¹. Briefly, 2 × 10⁴ yeast cells/well were used to infect A-431 cells in 96-well plates at 37 °C and 5% CO₂. The supernatants were carefully aspirated at 24 hpi, and cells were dried for 1 h. Then the cells were covered with 150 µL of 1% CV solution (Sigma) and incubated for 45 min at room temperature. The CV solution was aspirated, and the samples were washed with double-distilled water (H₂O_dd). The bound CV was liberated through incubation with 99% EtOH (Roth) for 45 min. 100 µL of the destained solution was transferred to a fresh 96-well plate and absorbance was measured at 550 nm using a microplate reader (Tecan Infinite M-Plex; i-control software).

To visualize the biofilm of the C. albicans strains, A-431 cells were seeded in 8-well µ-slides (ibidi GmbH) at 2 × 10⁴ cells/well. A-431 cells were cultured in RPMI-1640 supplemented with 10% FBS, for 2 days at 37 °C and 5% CO₂. After 2 days, the medium was removed and replaced with prewarmed fresh RPMI-1640 with or without 10 mg/mL albumin. The cells were infected with C. albicans SC5314 or 101 at a MOI of 5 for 24 h at 37 °C and 5% CO₂. After infection, infected cells were fixed with Histofix 4% (Carl Roth) for 15 min at RT. Subsequently, Histofix was washed away, replaced with PBS and stored for immunofluorescence staining at 4 °C.

For the immunofluorescence staining, samples were blocked and permeabilized (PBS with 1% BSA and 0.05% saponin) for 30 min at RT. C. albicans was stained using rabbit anti-C. albicans antibody (1:600 in PBS with 0.1% BSA and 0.1% saponin, rabbit anti-Candida, BP1006, Acris Antibodies) overnight, washed with PBS, and incubated with a secondary goat anti-rabbit AlexaFluor488 (1:500 in PBS with 0.1% BSA and 0.1% saponin, Invitrogen A32731) for 1 h in the dark at RT. PBS was added at the end of the staining to each condition.

Imaging of the biofilms was performed using a LSM 780 Zeiss confocal laser scanning microscope (Carl Zeiss Microscopy). Images were recorded using a 10× NA0.45 air immersion objective lens. A series of images was acquired at 4.51 μm intervals along the z-axis, allowing for a three-dimensional reconstruction of the biofilms from the start to the end of the fluorescent signal. The z-stack images were subsequently exported to the Napari interface for further analysis¹⁰².

Statistics and reproducibility

Data from in vitro epithelial cytotoxicity, fungal proliferation, 13-HODE release, individual metabolites and fungal respiration assays were analyzed using GraphPad Prism version 10 (GraphPad Software, La Jolla California USA). Data from at least three biological replicates were analyzed and values are presented as mean ± standard error of the mean (SEM). All microscopy analysis where at least independently repeated 3 times and representative images are provided in the manuscript. Used statistical tests are indicated in each figure legend. Statistical significance is depicted in the figures as P values. All data used to generate the graphs are provided as source data.

RNA isolation & analysis

Fungal RNA sample collection and isolation were carried out as previously described¹⁰³.

Before infection, the spent medium in the 6-well plate was exchanged with prewarmed fresh RPMI-1640 with or without 5 mg/mL albumin (Sanquin Plasma Products B.V). For mRNA expression analysis samples were obtained at 30 min, 3 h and 24 h incubation in RPMI-1640 with or without 5 mg/mL albumin at 37 °C and 5% CO₂. To obtain sufficient RNA yield A-431 cells were infected with the WT (SC5314; 6 × 10⁷ yeast cells/well) for the 30 min timepoint, whereas for the 3 h and 24 h time points, A-431 cells were infected with of 3 × 10⁶ yeast cells/well.

After incubation, the samples were centrifuged at 250 × g and 4 °C for 1 min, the supernatant was discarded, and 500 µL of RNeasy Lysis (RLT) buffer (Qiagen), containing 1% β-mercaptoethanol (Roth), were added. The well content was quickly detached using a cell scraper and collected in microcentrifuge tube (Sarstedt), directly snap-frozen in liquid nitrogen, and stored at –80 °C until further use. Collected samples were defrosted on ice, centrifuged at 20,000 × g for 10 min. The supernatant, containing most of the host RNA, was not used in this study. The fungal RNA was isolated from the remaining pellet. First, the pellet was resuspended in AE buffer 50 mM Na-acetate pH 5.3, 10 mM EDTA, and 1% SDS). After the addition of phenol/chloroform/isoamyl alcohol (25:24:1), the fungal cells were lysed using a freeze-thawing method. In brief, the samples were first transferred to a 65 °C water bath (GFL) for 5 min, before being transferred to –80 °C for 5 min. The process was repeated one more time. After the final heat-shock at 65 °C the samples were centrifuged at 20,000 × g and 4 °C for 10 min. The aqueous phase was transferred to a microcentrifuge tube. RNA was precipitated by incubating the samples with 500 µL of ice-cold isopropyl alcohol (Roth) and 100 µL cold sodium acetate (3 M, pH 5.5) for 1 h at –20 °C. Precipitated fungal RNA was collected by centrifugation at 20,000 × g at 4 °C for 15 min. The supernatant was discarded, and the RNA pellet was washed three times with 1 mL of ice-cold 70% ethanol (Roth). The RNA pellet was dried at 40 °C using a heating block (Eppendorf) and resuspended in 40 µL RNase-free water (Promega). RNA concentrations were quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and RNA quality was determined using a Bioanalyzer Agilent 2100 (Agilent Technologies). Samples were stored at –80 °C until further use. RNA sequencing was performed at Genewiz (Leipzig, Germany).

RNA-Seq bioinformatics analysis

The raw sequencing data was quality checked using FastQC v. 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC v. 1.12 software¹⁰⁴. Considering that the main quality control metrics such as Phred quality scores, GC content, and the adapter content showed high quality results, the step of read trimming was omitted. Further down-stream analysis of read mapping and quantification was performed as previously described¹⁰³. The data of C. albicans grown were mapped to the C. albicans SC5314 reference genome and genome annotations obtained from Candida Genome Database (CGD, last accessed on July 2022)¹⁰⁵. Only haplotype A of the phased C. albicans genome was used for this analysis in order to avoid high proportions of ambiguous mappings between alternative haplotypes. C. albicans GFF genome annotation file was converted to GTF format using gffread utility v. 0.9.8¹⁰⁶. The data of C. albicans samples grown with host cells were mapped to the concatenated reference genomes of C. albicans and human host. The primary assembly of human reference genome v. GRCh38.107 and corresponding annotations were obtained from Ensembl database (last accessed in June 2022)¹⁰⁷. Read mapping and gene-level read count quantification was performed by spliced-junction-aware read mapping software STAR v. 2.7.10a¹⁰⁸. Potential read cross-mapping between human and C. albicans genomes was assessed with Crossmapper v. 1.1.1¹⁰⁹. Differential gene expression analysis was performed using the Bioconductor package DESeq2 v. 1.42.0¹¹⁰ using read counts obtained by STAR. We compared the albumin treated to non-albumin treated samples for all time points (0.5 hpi, 3 hpi and 24 hpi) by Wald test using the contrast option of DESeq2. Genes with |log₂ fold change | >1 and adjusted P value (P_adj) <0.01 were considered differentially expressed, unless specified otherwise. Possible batch effects were inspected using Principal Component Analysis (PCA). Gene Ontology (GO) term enrichment analysis was performed using clusterProfilerpackage v. 4.10.0¹¹¹. Adjustment of P values was done by Benjamini-Hochberg procedure. GO information for C. albicans was obtained from CGD and visualized in R. All custom calculations and visualizations were performed in R v. 4.3.2 using various packages.

Metabolome sampling and analysis

For untargeted metabolomics, C. albicans (SC5314) was cultured in 150 cm² culture flasks (Sarstedt AG & Co. KG) at 2 × 10⁹ yeast cells/flask for 3 h, and 5 × 10⁷ yeast cells/flask for 24 h in RPMI-1640 culture medium with or without 5 mg/mL albumin at 37 °C and 5% CO₂. To assess the exometabolome, supernatants were collected at 24 h as a control uninfected culture medium was collected after 3 h and 24 h incubation without albumin. For each condition 1 mL supernatant was collected from the 150 cm² flask, centrifuged at 10,000 × g and 4 °C for 1 min, 500 µL were transferred into a 1.5 mL collection tube, snap-frozen in liquid nitrogen, and stored at –80 °C until analysis. For assessment of the intracellular metabolome the remaining supernatants were removed, the fungal biofilm was washed with ice-cold PBS, scraped, collected in a 50 mL tube, and centrifuged at 10,000 × g and 4 °C for 5 min. After another washing step the supernatant was discarded, the cell pellet was collected in a 1.5 mL collection tube, snap-frozen in liquid nitrogen, and stored at –80 °C until analysis. All collection steps were carried out on ice. Untargeted metabolomics was performed by Metabolon (Morrisville, US). For a detailed description please refer to the Supplementary methods.

Metabolome statistical analysis

For analysis, the raw metabolome data was rescaled to set the median of all metabolites equal to 1, the missing values were imputed with the minimum, and each value was transformed using the natural log. Thus, the abundance of individual metabolites is normalized to the median abundance of all metabolites. The heatmaps were generated by loading the data in R-studio version 2023.03.0, rows were normalized, and Euclidian distances were calculated using the pHeatmap package 1.0.12. The clusters were manually obtained. The complete linkage agglomeration method was used to get a hierarchical cluster analysis of the distance matrix of each metabolite. Color bars indicating experimental conditions or cluster group were added using the R package dendextend 1.17.1. To generate the PCA the R function prcomp (v 4.2.1) was used. The graphs were generated using the ggbiplot package v 0.55. Volcano plots were analyzed using GraphPad Prism version 10. The annotated KEGG metabolic pathways map 01100³⁵ was created using iPath3.0¹¹².

Fungal respiration measurement

Washed C. albicans (SC5314 and 101) overnight cultures were seeded in 200 µL RPMI-1640 for Seahorse (Agilent Technologies, Santa Clara, CA), supplemented with 2 mM l-Glutamine, 1 mM sodium pyruvate and 11 mM d-Glucose with or without 10 mg/mL albumin (Sanquin Plasma Products B.V.) as 10⁴ yeast cells/well in a Seahorse 96-well cell culture plate. Fungi were incubated for 4 h at 37 °C w/o CO₂ allowing rigid attachment to the plate. Afterwards, cells were carefully washed once and the medium was exchanged with Seahorse RPMI-1640 including mentioned supplements, without albumin. The plate was incubated for further 30 min at 37 °C without CO₂ for equilibration and then inserted into a Seahorse XFe96 Analyzer (Agilent Technologies) loaded with a hydrated cartridge, for measurement. As measurement protocol, a Mitostress-based procedure was chosen with the basal fungal respiration averaged from four consecutive measurement cycles every 6 min.

Metabololipidomics

For oxylipin analysis, 200 µL culture supernatants were transferred to 2 mL of ice-cold methanol containing 10 µL of deuterium-labeled internal standards (200 nM d8-5S-HETE, d4-LTB₄, d5-LXA₄, d5-RvD₂, d4-PGE₂ and 10 µM d8-AA; Cayman Chemical/Biomol GmbH, Hamburg, Germany) to facilitate quantification and sample recovery. Sample preparation was conducted by adapting published criteria¹¹³. In brief, samples were kept at −20 °C for 60 min to allow protein precipitation. After centrifugation (1200 × g, 4 °C, 10 min) 9 mL acidified H₂O was added (final pH = 3.5) and samples were subjected to solid phase extraction. Solid phase cartridges (Sep-Pak® Vac 6cc 500 mg/ 6 mL C18; Waters, Milford, MA) were equilibrated with 6 mL methanol and 2 mL H₂O before samples were loaded onto columns. After washing with 6 mL H₂O and additional 6 mL n-hexane, oxylipins were eluted with 6 mL methyl formate. Finally, the samples were brought to dryness using an evaporation system (TurboVap LV, Biotage, Uppsala, Sweden) and resuspended in 100 µL methanol-water (50/50, v/v) for UPLC-MS/MS automated injections. Oxylipin profiling was analyzed with an Acquity™ UPLC system (Waters, Milford, MA, USA) and a QTRAP 5500 Mass Spectrometer (ABSciex, Darmstadt, Germany) equipped with a Turbo V™ Source and electrospray ionization. Oxylipins were eluted using an ACQUITY UPLC® BEH C18 column (1.7 µm, 2.1 × 100 mm; Waters, Eschborn, Germany) at 50 °C with a flow rate of 0.3 mL/min and a mobile phase consisting of methanol-water-acetic acid of 42:58:0.01 (v/v/v) that was ramped to 86:14:0.01 (v/v/v) over 12.5 min and then to 98:2:0.01 (v/v/v) for 3 min¹¹⁴. The QTrap 5500 was operated in negative ionization mode using scheduled multiple reaction monitoring (MRM) coupled with information-dependent acquisition. The scheduled MRM window was 60 sec, optimized oxylipin parameters were adopted¹¹⁵, and the curtain gas pressure was set to 35 psi. The retention time and at least six diagnostic ions for each oxylipin were confirmed by means of an external standard (Cayman Chemical/Biomol GmbH, Hamburg, Germany). Quantification was achieved by calibration curves for each oxylipin. Linear calibration curves were obtained for each oxylipin and gave r² values of 0.998 or higher (for fatty acids 0.95 or higher). Additionally, the limit of detection for each targeted oxylipin was determined.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Acknowledgements

S.U.J.H., C.F.F., and M.S.G. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Emmy Noether Program (project no. 434385622 / GR 5617/1-1) to M.S.G. S.U.J.H. was additionally supported by the Jena School for Microbial Communication with funding from the Carl Zeiss Stiftung to M.S.G. A.D. was supported by an Exploration Grant of the Boehringer Ingelheim Foundation (BIS) to M.S.G. This work further was supported by the FWO-funded SBO project DeVEnIR (project number S006424N) to M.S.G. B.C., M.P., M.S.G., K.G., P.M.J., O.W., S.V., B.R., and J.M. were further supported by the DFG within the Collaborative Research Center (CRC)/Transregio (TRR) 124 FungiNet projects A7, C1 and C2 (DFG project number 210879364). SV was further supported by the German Ministry for Education and Science in the program Unternehmen Region (BMBF 03Z22JN11). S.A. was supported by funding from the European Union’s Horizon 2020 research and innovation program under grant agreement 847507 (HDM-FUN). This research was supported by the Free State of Thuringia and co-funded by the European Union—Project-ID 2023 FGI 0004—A Live broadcast of the interactions between host and fungal pathogens. A.D. was supported by a scientist exchange grant from the excellence cluster EXC 2051 Balance of the Microverse—Project-ID 390713860 funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy. We acknowledge Salomé LeibundGut-Landmann (Section of Immunology, Vetsuisse-Faculty, University of Zurich) for providing the 101 strain. We acknowledge the laboratory of Experimental Internal Medicine (Radboudumc, Nijmegen) for the possibility to perform respiration measurements. We acknowledge Mart Sillen (KU Leuven) for providing protocols for microscopic biofilm analysis. We acknowledge Bernhard Hube (Microbial Pathogenicity Mechanisms, Leibniz-HKI) for fruitful discussions and providing access to deletion mutants. We thank Stephanie Wisgott, Nadja Jablonowski (Microbial Pathogenicity Mechanisms, Leibniz-HKI), Nikshitha Tulasi, Ceren Oktay (Adaptive Pathogenicity Strategies, Leibniz-HKI), and Kevin Schlabach (Paleobiotechnology, Leibniz-HKI) for technical support.

Author contributions

S.U.J.H.: Investigation, Formal analysis, Visualization, Writing—Original Draft, Writing—Review & Editing; C.F.F.: Investigation, Validation, Formal analysis, Visualization, Writing—Review & Editing; K.G.: Investigation, Formal analysis, Visualization, Writing—Review & Editing; A.D.: Investigation, Formal analysis, Visualization, Writing—Review & Editing, Supervision; H.H.: Formal analysis, Data Curation, Visualization, Writing—Review & Editing; A.M.: Investigation, Visualization S.A.: Investigation; B.C.: Investigation, Visualization; G.V.: Investigation; T.Z.: Investigation; M.P.: Investigation; B.R.Z.: Resources; J.M.: Resources, Methodology, Writing—Review & Editing, Supervision, Funding acquisition; O.W.: Methodology, Writing—Review & Editing, Supervision, Funding acquisition; T.G.: Methodology, Writing—Review & Editing, Supervision P.M.J.: Investigation, Writing—Review & Editing, Supervision S.V.: Resources, Conceptualization, Writing—Review & Editing, Funding acquisition M.S.G.: Conceptualization, Methodology, Formal analysis, Visualization, Writing—Original Draft, Writing—Review & Editing, Supervision, Project administration, Funding acquisition.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Data availability

The data supporting the findings of this study and metadata to reproduce experiments are available within the paper and its supplementary information. All relevant data are available by request. The transcriptomics data generated in this study have been deposited in the Sequencing Read Archive (SRA) database under accession code PRJNA1132138. The processed transcriptome data generated in this study are provided in the Supplementary Information. The untargeted metabolomics data generated in this study are provided in the Supplementary Information.  are provided with this paper.

Code availability

All codes, read count data and used packages and their versions are available at [https://github.com/Gabaldonlab/C_albicans_with_albumin] for result reproducibility.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41467-025-61701-5.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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