The Candida albicans exotoxin candidalysin promotes alcohol-associated liver disease

Abstract

Background & aims: Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease.

Methods: C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin.

Results: The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the β-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis.

Conclusions: Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis.

Lay summary: Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the β-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.

Keywords: Alcohol-related liver disease; Microbiome; Microbiota; Mycobiome.

Conflict of interest statement

Conflict of interest: B.S. is consulting for Ferring Research Institute.

Published by Elsevier B.V.

Figures

Fig. 1.. Candidalysin is related with the development of alcoholic liver disease.

Fecal samples from controls (n=11), patients with alcohol use disorder (AUD) (n=42) and alcoholic hepatitis (n=91) were cultured on YPD agar plates with antibiotics. Each colony was then assessed by qPCR to confirm as fungus, and to determine positivity for C. albicans or ECE1. (A) Colony forming units (CFUs) of total fungi in fecal samples. (B) Colony forming units (CFUs) of C. albicans in fecal samples. (C) Percentage of subjects with fecal samples positive for fungi. (D) Percentage of subjects with fecal samples positive for C. albicans. (E) Percentage of subjects with fecal samples positive for ECE1. Results are expressed as mean ± s.e (A and B). P values were determined by Kruskal-Wallis test with Dunn’s post-hoc test (A and B), and Z test followed by false discovery rate (FDR) procedures (C - E). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 2.. Effects of Candidalysin on ethanol-induced liver disease.

C57BL/6 mice were fed an oral isocaloric (control) diet (1–2 technical replicates) or chronic-plus-binge ethanol diet (3–4 technical replicates), and gavaged with vehicle (PBS), wild type C. albicans (Wild type), ECE1 deleted C. albicans (ece1Δ/Δ), only Ece1-III62–93 deleted C. albicans (ece1Δ/Δ + ECE1Δ184–279) or ECE1 re-integrant C. albicans (ece1Δ/Δ + ECE1) with an amount of 108 colony forming units (CFUs) every third day. (A) Serum levels of alanine aminotransferase (ALT). (B) Hepatic triglycerides levels. (C) Representative images of Oil Red O stained liver tissue. (D) Hepatic levels of Il1b mRNA. (E) Hepatic levels of Cxcl1 mRNA. (F) Hepatic levels of Cxcl2 mRNA. (G) Serum levels of ethanol in mice fed chronic-plus-binge ethanol diet. (H) Hepatic levels of Adh1 mRNA. (I) Hepatic levels of Cyp2e1 mRNA. (Control diet: PBS, n=8; Wild type, n=5; ece1Δ/Δ, n=5; ece1Δ/Δ + ECE1Δ184–279, n=5; ece1Δ/Δ + ECE1, n=5; Ethanol diet: PBS, n=14; Wild type, n=14; ece1Δ/Δ, n=14; ece1Δ/Δ + ECE1Δ184–279, n=14; ece1Δ/Δ + ECE1, n=12). Scale bars = 100 μm. Images were taken at ×100 magnification. Results are expressed as mean ± s.e (A, B, D - I). P values were determined by one-way ANOVA with Tukey’s post-hoc test (A, B, D - I). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 3.. Effects of Candidalysin on ethanol-induced liver disease in mice lacking CLEC7A in bone marrow derived cells.

C57BL/6 mice underwent transplantation of Clec7a−/− bone marrow (Clec7aΔBM) and were fed an oral chronic-plus-binge ethanol diet (3 technical replicates). Mice were gavaged with vehicle (PBS), wild type C. albicans (Wild type) or ECE1 deleted C. albicans (ece1Δ/Δ) with an amount of 108 CFUs every third day. (A) Serum levels of ALT. (B) Hepatic triglycerides levels. (C) Representative images of Oil Red O stained liver tissue. (D) Hepatic levels of Il1b mRNA. (E) Hepatic levels of Cxcl1 mRNA. (F) Hepatic levels of Cxcl2 mRNA. (G) Serum levels of ethanol. (H) Hepatic levels of Adh1 mRNA. (I) Hepatic levels of Cyp2e1 mRNA. (PBS, n=10; Wild type, n=11; ece1Δ/Δ, n=11). Scale bars = 100 μm. Images were taken at ×100 magnification. Results are expressed as mean ± s.e. (A, B, D - I). P values were determined by one-way ANOVA with Tukey’s post-hoc test (A, B, D - I). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 4.. Candidalysin does not affect intestinal permeability in mice fed ethanol.

C57BL/6 mice were fed an oral isocaloric (control) diet (1–2 technical replicates) or chronic-plus-binge ethanol diet (3–4 technical replicates) and gavaged with vehicle (PBS), wild type C. albicans (Wild type) or ECE1 deleted C. albicans (ece1Δ/Δ) with an amount of 108 CFUs every third day. (A) Serum levels of FD4. Mice were gavage-fed FITC labeled dextran (200μl, 100mg/ml) 1 hour after binge on the last day and then sacrificed 4 hours later, and fluorescence was measured in the serum. (Control diet: PBS, n=6; Wild type, n=6; ece1Δ/Δ, n=5; Ethanol diet: PBS, n=5; Wild type, n=6; ece1Δ/Δ, n=6). (B) Serum levels of intestinal fatty-acid binding protein (IFABP) (Control diet: PBS, n=7; Wild type, n=5; ece1Δ/Δ, n=5; Ethanol diet: PBS, n=14; Wild type, n=13; ece1Δ/Δ, n=10). Results are expressed as mean ± s.e (A and B). P values were determined by two-way ANOVA with Tukey’s post-hoc test (A and B). ****P<0.0001.

Fig. 5.. Candidalysin induces death of cultured primary hepatocytes.

Primary hepatocytes were isolated from mice fed an oral isocaloric (control) diet (A and C) or from mice subjected to the chronic-plus-binge ethanol diet model (B and D). Hepatocytes were incubated with different concentrations (0.6 μM and 3 μM) of Candidalysin or negative control peptide (Ece1-VII), as well as blank control (equal volume of culture medium), without (−) or with (+) ethanol (25 mM) for 24 hours (3 independent experiments performed in 4–7 replicates). (A and B) Cytotoxicity was determined by measuring LDH release in the supernatant. (C and D) Hepatocyte survival was measured using the MTT assay. Results are expressed as mean ± s.e. P values were determined by two-way ANOVA with Tukey’s post-hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 6.. Presence of Candidalysin associates with disease severity and mortality in patients with alcoholic hepatitis.

(A) MELD score for patients with alcoholic hepatitis. (Candidalysin positive, n=27; Candidalysin negative, n=62). (B) Kaplan-Meier curve of 90-day mortality for patients with alcoholic hepatitis. (Candidalysin positive, n=25; Candidalysin negative, n=61). Patients were grouped according to their ECE1 status in stool. Patients lost to follow-up were censored at the time they were last seen alive. Results are expressed as median with range. P value was determined by Mann-Whitney-Wilcoxon test (A) or Log-rank test (B).