Lactobacillus rhamnosus colonisation antagonizes Candida albicans by forcing metabolic adaptations that compromise pathogenicity

Abstract

Intestinal microbiota dysbiosis can initiate overgrowth of commensal Candida species - a major predisposing factor for disseminated candidiasis. Commensal bacteria such as Lactobacillus rhamnosus can antagonize Candida albicans pathogenicity. Here, we investigate the interplay between C. albicans, L. rhamnosus, and intestinal epithelial cells by integrating transcriptional and metabolic profiling, and reverse genetics. Untargeted metabolomics and in silico modelling indicate that intestinal epithelial cells foster bacterial growth metabolically, leading to bacterial production of antivirulence compounds. In addition, bacterial growth modifies the metabolic environment, including removal of C. albicans' favoured nutrient sources. This is accompanied by transcriptional and metabolic changes in C. albicans, including altered expression of virulence-related genes. Our results indicate that intestinal colonization with bacteria can antagonize C. albicans by reshaping the metabolic environment, forcing metabolic adaptations that reduce fungal pathogenicity.

Fig. 1. Reduced C. albicans inocula cause significant damage compared to C. albicans in the presence of live L. rhamnosus that reduces pathogenicity contact-independently.

a Fungal burden assessed by quantification of C. albicans CFUs and b the necrotic cell damage of IECs quantified by LDH activity in supernatants at 24 h post infection (hpi). Cells were infected with a C. albicans infection inoculum (4 × 10⁵/ml) in the presence and absence of L. rhamnosus, or with a reduced infection inoculum (1 × 10⁴/ml) in the absence of L. rhamnosus (n = 3 biological repeats; L. rhamnosus colonized A * = p 0.0276, B * = p 0.0434 reduced inoculum A * = p 0.0225; B * = p 0.0434). c, d Necrotic cell damage of IECs quantified by LDH activity in supernatants at 24 hpi, (c) in the presence and absence of L. rhamnosus colonization and antibiotic treatment with Gentamicin and Penicillin/Streptomycin at 4 hpi (n = 4 biological repeats; * = p 0.0173 ns = 0.184), or (d) in the presence and absence of L. rhamnosus colonization where L. rhamnosus was in direct contact with the cells or physically separated using a transwell insert with a 0.4 µm pore size (n = 3 biological repeats; *** = p 0.0009, * = 0.048). Bars represent the mean and standard deviation of the independent experiments, dots represent the mean of the technical replicates of the individual experiments, biological repeats were compared for significance using an unpaired t-test (two-tailed, one-sample), * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001. Source data are provided as a Source Data file.

Fig. 2. IECs metabolically foster L. rhamnosus growth.

a Principal component analysis of metabolite composition assessed by untargeted metabolomics in supernatants collected at 6 and 12 hpi. Arrows (I, II, III) indicate shifts in the metabolic profile. b Differentially increased or decreased metabolites in comparison of the different conditions shown in proportional Venn diagrams. Data summarized from n = 5 at 6 and 12 hpi. c Hierarchical clustering based on Euclidean distance of relative metabolite abundance in the supernatants at 6 hpi. d Growth of L. rhamnosus in KBM, on IECs, in transwells physically separated from IECs, or in supernatants of IECs (spent) assessed by counting CFUs on MRS agar. Data are shown as the mean and standard deviation (SD) of n = 3 biological replicates. e In silico prediction of L. rhamnosus biomass formation in KBM or supernatants of IECs. f Growth of L. rhamnosus assessed by CFUs after 48 h incubation in KBM supplemented with single metabolites or combinations of metabolites. Data of n = 3–6, KBM = 17, IEC spend n = 8 biological replicates are shown as the mean and SD. Data were tested for significance using a t test (two tailed, one-sample) against growth in KBM, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001 (CA + GGA + CARN n = 6, * = p 0.0410; CA + CARN n = 5, **** = p < 0.0001; CA + GGA n = 3, *** = p 0.0003; CA + OB n = 3, ** = p 0.0041; CA n = 5, *** = p 0.0003; CA + OV n = 3, ** = p 0.0089, CARN n = 4, * = p 0.0218; GGA + CARN n = 3, *** = p 0.0001, CA + NAG n = 3, **** = p < 0.0001). Source data are provided as a Source Data file. g Comparison of metabolic pathway activity levels between different conditions as indicated. Relative pathway change was determined by identifying the number of pathway-specific reactions for which feasible flux ranges differ according to flux variability analysis.

Fig. 3. L. rhamnosus induces an unfavourable environment for C. albicans.

a, b Representative images of C. albicans morphology following growth in presence of (a) different metabolites at 50 mM, at neutral or acidic pH, for 20 h at 37 °C with 5% CO₂ (n = 2 biological replicates) or (b) a combination of selected metabolites each at 5 mM (cytosine, phenylpyruvate, 2-hydroxy-4-(methylthio)-butyric acid, 3-phenyllactic acid, 2-Hydroxyisocaproic acid and d-indole-3-lactic acid) and lactic acid at 15 mM, for 20 h at 37 °C with 5% CO₂ (n = 4 biological replicates). Yeast morphology is indicated with arrowheads. c Hyphal length of C. albicans grown for 4 h in KBM in the presence or absence of the combination of selected metabolites at 37 °C with 5% CO₂ (n = 400 cells examined over 4 independent experiments) (* = p 0.0108). d Necrotic damage of IECs measured by the LDH activity in the supernatant at 24 hpi with C. albicans in the presence or absence of the combination of selected metabolites (** = p 0.0093). e C. albicans-induced necrotic damage of IECs measured by the LDH activity in the supernatant (* = p 0.0385) and (f) C. albicans translocation across the epithelial barrier assessed in the presence or absence of cytosine at 50 mM at 24 hpi (* = p 0.0386). Bars represent the mean and SD of n = 4 independent experiments, dots represent the mean of the technical replicates of the individual experiments, boxplots represent the distribution of the total measurements (centre line, median; box limits, upper and lower quartiles; whiskers, range). Biological repeats were compared for significance using paired t tests (two-tailed, one-sample) on the means of the technical replicates. Source data are provided as a Source Data file. g Phenotypic microarray growth experiments for wild-type C. albicans in presence of each metabolite as a carbon, nitrogen, or phosphorous source (left), metabolome data measured at 6 and 12 h and metabolic modelling predictions (right) are indicated for selected metabolites. For metabolic modelling, media was adapted from metabolome data derived from supernatants of IECs. Uptake or secretion was determined by identifying feasible flux ranges for metabolite-specific exchange reactions alongside optimization for biomass. Asterisks show statistical significance. ANOVA was performed for phenotypic microarrays (two-sided), Wilcoxon test for metabolomics (two-sided), with FDR correction. * = p ≤ 0.05). For the entire panel of metabolites see Fig. S5.

Fig. 4. C. albicans undergoes transcriptional changes during infection of L. rhamnosus-colonized IECs.

a Principal component analysis of C. albicans gene expression at 6 and 24 h during in vitro infection of IECs in the presence and absence of L. rhamnosus colonization. b Volcano plots showing differentially regulated C. albicans genes at 6 and 24 hpi as a result of L. rhamnosus colonization prior to infection based on the criteria of a Log₂ fold change of >1 or < −1 and a Bonferroni-corrected two-tailed moderated t-test p-value of <0.05 (dark blue and dark red) and <0.1 (light blue and light red). Source data are provided as a Source Data file. c Venn diagram analysis of the overlap in differentially expressed genes at 6 and 24 hpi. Data summarized from n = 3 and n = 4 independent experiments at 6 and 24 hpi, respectively. d PCA of C. albicans gene expression at 24 hpi during in vitro infection of IECs in the presence and absence of L. rhamnosus colonization and in the presence and absence of antibiotics.

Fig. 5. C. albicans undergoes transcriptional metabolic adaptations when infecting L. rhamnosus colonized epithelium.

a GO-term enrichment analysis of differentially regulated genes (Log₂ fold change >1 or < −1 and p < 0.1) analysed with the GO-term finder on the Candida Genome Database website and reduced with the Revigo program (http://revigo.irb.hr/) (similarity: Tiny (0.4)). Significantly enriched GO terms are plotted based on the –Log₁₀ p-value from the Bonferroni-corrected hypergeometric distribution. Data summarized from n = 3 at 24 hpi. Source data are provided as a Source Data file. b Heatmap highlighting the transcriptional regulation of C. albicans metabolic genes as a result of L. rhamnosus colonization at 6 and 24 hpi. Legend colour represents the Log₂ fold change of the regulation in presence vs. absence of L. rhamnosus. The asterisks (*) represent significance, based on the criteria of a Log₂ fold change of >1 or < −1 and a moderated t-test, Bonferroni-corrected two-tailed p-value of <0.05.

Fig. 6. Central metabolism of C. albicans is altered by L. rhamnosus colonization.

Reactions associated to glycolysis, TCA cycle, oxidative phosphorylation are indicated, as well as relationships with additional metabolic pathways (pentose phosphate pathway, nitrogenated bases, sulfur metabolism, butanoate, propanoate, and pantothenate, CoA, β-Alanine and glutathione metabolism). Information on metabolome (12 h) and transcriptomic (24 h) data are combined with in silico genome-wide metabolic flux predictions (12 h). Dotted arrows represent several combined reactions. For the reaction abbreviations see Table S3.

Fig. 7. Screening of C. albicans deletion mutants.

a Ability of deletion mutants to induce necrotic cell damage of IECs assessed by LDH activity in the supernatant at 24 hpi. Data are shown as the mean and standard deviation (SD) with dots showing the individual replicates (n = 3–11 biological replicates). Deletion mutants were compared to the wild-type control using a one-way ANOVA and Dunnett’s Multiple Comparison post-hoc analysis. Mutants with a significantly increased or decreased damage potential (dark blue, p-value ≤ 0.05; light blue, p-value ≤ 0.1) are labelled (see Supplementary Table 4 for exact p-values). Horizontal lines correspond to the mean of the damage induced by the wild-type ± SD. b Growth rates of the ptp3Δ/Δ mutant with significantly reduced growth (blue line) compared to the parental strain (black line) in KBM. Lines represent the mean and SD of n = 3 independent experiments and were compared for significance using a two-way Repeated Measures ANOVA, * = p 0.0188. Source data are provided as a Source Data file. c Representative images of the ptp3Δ/Δ deletion mutant and the parental strain morphologies, after 24 h incubation in KBM at 37 °C with 5% CO₂.