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.

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.

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.

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 formation. 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.

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, G n = 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.

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.

Fig. 6. Albumin rewires metabolic pathways in C. albicans.

A Differential changes in intracellular metabolite abundance plotted on the KEGG metabolic pathways map (01100) using iPath3. 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.

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.

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.