Impaired amino acid uptake leads to global metabolic imbalance of Candida albicans biofilms

Abstract

Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development.

Fig. 1. Transcriptional responses in aging biofilms affect various metabolic processes.

Differentially regulated genes (log₂FC ± 1; p < 0.05) in late biofilms were compared to earlier time points (24 vs. 8 h; 48 vs. 8 h; 48 vs. 24 h) and GO terms for molecular processes were each summarized using REVIGO with a cutoff of 0.5 dispensability. Metabolism-related processes were separated from others. Up-regulated terms are shown in green, and down-regulated terms are in red. Source data are provided as a Source Data file.

Fig. 2. Intracellular and extracellular metabolomics in aging biofilms.

Differentially abundant metabolites from each metabolite family were counted and plotted as a heat map. The extent of the metabolic changes is color-coded with darker fillings for bigger changes. The scale bar indicates the metabolite counts within one metabolite family. No significant changes are marked in gray. Cutoff p < 0.05 and log₂FC ± 1. Source data are provided as a Source Data file. a The intracellular metabolite content increased after 24 h, but after 48 h of biofilm formation, many metabolites decreased significantly. The largest decline was observed for the nitrogenous substrates (amino acids and nucleotides). b Many metabolites accumulated in the extracellular metabolome in mature biofilms as amino acids and nucleotides were most abundant at the late time point.

Fig. 3. Secretome analysis of mature C. albicans biofilm media.

a Volcano plot illustrating the log₂ ratio of secreted proteins in 48 and 24 h biofilms to the corresponding p_adj value (−log₁₀). Dashed lines symbolize the cutoff (p < 0.05 and log₂FC ± 2). A total of 1103 different proteins were identified in all biofilms examined, with 801 proteins detected at 24 h and 1067 proteins detected at 48 h. Only 14% contained a signal peptide (SP) for directed secretion. Both the fraction with and without SP showed higher protein quality after 48 h. b GO term analysis of all detected proteins containing an SP. Counts represent observed protein abundance compared to the number of proteins assigned to the term (size). Results for molecular function and biological process were each summarized using REVIGO with a cutoff of 0.5 dispensability. Source data are provided as a Source Data file.

Fig. 4. Principal component analyses for transcriptome and metabolome data sets from maturing wild-type biofilms.

Gene expression pattern from 8 h normoxic biofilms builds a distinct group, whereas older normoxic biofilms cluster together with hypoxic biofilms from all time points. Intra- and extracellular metabolomes strongly cluster by the oxygen state and show a mostly time-point-independent distribution. Source data are provided as a Source Data file.

Fig. 5. GO term analysis of differentially expressed genes in 8 h wild-type biofilms grown under hypoxia vs. normoxia.

Enriched GO terms were determined for hypoxia up-regulated genes (741 total) and hypoxia down-regulated genes (488 total) in contrast to the normoxic counterparts. Count describes the number of measured differentially regulated genes of a process (cluster frequency) and size measures the genomic background frequency. All GO terms were summarized using REVIGO with stringent settings (Cutoff: 0.7 dispensability). Source data are provided as a Source Data file.

Fig. 6. Intracellular and extracellular metabolomics in hypoxic vs. normoxic biofilms.

Metabolomes from cells and media from hypoxic biofilms were compared to normoxic biofilms and numbers of significant changes (cutoff p < 0.05 and log₂FC ± 1) were plotted for main metabolic pathways. Numbers of differentially abundant metabolites were analyzed and summarized according to the respective pathways for (a) 8 h, (b) 24 h, and (c) 48 h biofilms. Significant changes (log₂FC) in the abundance of intracellular (d) and extracellular amino acids (e) were plotted. Source data are provided as a Source Data file.

Fig. 7. Transcriptome analyses in stp2Δ biofilms compared to the wild type under normoxia.

Transcriptomes of stp2Δ and wild-type biofilms grown for 8, 24, and 48 h under normoxic conditions and differentially expressed genes (p < 0.05 and log₂FC ± 1) were analyzed for GO term enrichments and summarized with REVIGO (dispensability < 0.5 and p < 0.05 were displayed). Source data are provided as a Source Data file. a VENN diagram of time point-specific effects shows the biggest effects for stp2Δ at early time points. b Time point-independent (time point as a confounding variable) differences were analyzed for genotype-specific effects during biofilm formation.

Fig. 8. Comparative intracellular and extracellular metabolomics of stp2Δ mutant vs. wild-type biofilms.

Metabolomes from cells and media were analyzed for significant metabolic changes in stp2Δ mutant and wild-type biofilms. Analyzed contrasts were time point-adjusted. Numbers of metabolites with significant changes (Cutoff p < 0.05 and log₂FC ± 1) were plotted for main metabolic families (a) and amino acids subgroups (b). Metabolite data were time point-adjusted (time point as a confounding variable) before comparative analysis. c Box plot diagram shows relative abundance (log₂FC, median, upper and lower quartiles) of extracellular amino acids from wild-type (black) and stp2Δ (red) media, which were normalized to the respective amino acid concentrations from RPMI blank medium. Statistical differences between strains were calculated using the Student’s t-test and plotted next to the corresponding amino acid (* for p < 0.05). Source data are provided as a Source Data file.