Enterotypes of the human gut mycobiome

Abstract

Background: The fungal component of the human gut microbiome, also known as the mycobiome, plays a vital role in intestinal ecology and human health. However, the overall structure of the gut mycobiome as well as the inter-individual variations in fungal composition remains largely unknown. In this study, we collected a total of 3363 fungal sequencing samples from 16 cohorts across three continents, including 572 newly profiled samples from China.

Results: We identify and characterize four mycobiome enterotypes using ITS profiling of 3363 samples from 16 cohorts. These enterotypes exhibit stability across populations and geographical locations and significant correlation with bacterial enterotypes. Particularly, we notice that fungal enterotypes have a strong age preference, where the enterotype dominated by Candida (i.e., Can_type enterotype) is enriched in the elderly population and confers an increased risk of multiple diseases associated with a compromised intestinal barrier. In addition, bidirectional mediation analysis reveals that the fungi-contributed aerobic respiration pathway associated with the Can_type enterotype might mediate the association between the compromised intestinal barrier and aging.

Conclusions: We show that the human gut mycobiome has stable compositional patterns across individuals and significantly correlates with multiple host factors, such as diseases and host age. Video Abstract.

Keywords: Enterotype; Fungi; Gut mycobiome; ITS; Metagenomics.

Fig. 1

Composition and diversity of the human gut mycobiome across studies and geographic sites. a Geographic distribution of study populations and associated fungal enterotypes, where the datasets are sequenced with either ITS1 or ITS2 barcodes. b Genus-level gut mycobiome composition across the three continents (North America, Europe, and Asia). c Cumulative curves of the number of detected genera according to the number of sequenced samples from different study populations. d The distribution of fungal Shannon diversity across study populations. The Venn diagram shows the number of fungal genera detected by ITS1- and ITS2- based amplification. e, The correlation between the Shannon index of bacteria and that of fungi in the Zuo et al. [22] cohort, with shaded region representing 95% confidence intervals of the linear regression

Fig. 2

The enterotypes of the human gut mycobiome. a Clustering results of fungal enterotypes on ITS1 and ITS2 datasets and visualized by principal coordinate analysis (PCoA), and the most abundant genera within each enterotype is shown. The between-sample distances within each cluster compared to the median distance between clusters (black line) are shown at the bottom right of each panel. The bar height is the median distance, and the whiskers represent the 25th and 75th quantiles. b A four-enterotype classifier trained on the ITS2-sequencing datasets was applied to predict enterotypes in the ITS1-sequencing datasets, and the corresponding Area Under the Receiver Operating Characteristic Curve (AUC) values were presented. “Without drivers” refers to excluding the driver genera Candida, Saccharomyces, Aspergillus, Saccharomycetales sp. and Ascomycota sp. when training the classifiers. c The concordance of enterotype-associated fungal genera and enrichment trends across different cohorts, and log(FC) denotes the log-transformed fold change of the average relative abundance of the genera within respective enterotypes relative to that of others. The taxa name with a placeholder means that it could not be confidently assigned to a known taxonomic group. Asterisks represent the statistical significance of the multiple testing corrected one-sided Wilcoxon rank-sum tests (*adjusted p < 0.05, **adjusted p < 0.01, ***adjusted p < 0.001). d The correlations between fungal enterotypes and bacterial enterotypes in the CHGM cohort. The color reflects the O/E ratio (the ratio of observed count to expected count), and asterisks represent the statistical significance of Fisher’s exact test for each pair of comparison: *p < 0.05, **p < 0.01

Fig. 3

Age distribution and the gut aging indices of fungal enterotypes. a Age distribution of fungal enterotypes in two cohorts from China with p values from Wilcoxon rank-sum test p values shown for the age difference between enterotypes (left two panels). The right panel shows the proportion of fungal enterotypes in young (18–30 years), middle (31–60 years), and old (> –60 years) age groups from these two cohorts, respectively, with asterisks showing the statistical significance of multiple testing corrected Fisher’s exact test (*adjusted p < 0.05, ** adjusted p < 0.01, *** adjusted p < 0.001). b The age-associated fungal genera with p values < 0.05 determined by multivariate linear regression with adjustment of gender and cohort, where the red bar represents a positive correlation while the blue one represents a negative one. c The correlation between the gut aging index (GAI) and age after the LOESS smoothing for each fungal enterotype on four cohorts with available age data (CHGM cohort, Gao et al. [23], Limon et al. [12], and Zuo et al. [22]). Sacc_type: p = 2.1e − 03, Pearson’s r = 0.30; Can_type: p = 8.4e − 10, Pearson’s r = 0.45; Asp_type: p < 3.0e − 06, Pearson’s r = 0.36; Asc_type: p = 1.3e − 02, Pearson’s r = 0.27. d The distribution of GAI across fungal enterotypes in different cohorts. Wilcoxon rank-sum test p values are displayed above the boxplots

Fig. 4

Metabolic pathways associated with fungal enterotypes. a The fungal pathways enriched in different fungal enterotypes (bottom) and associated fungal genera (top). Log(FC) denotes log-transformed fold change of the average relative abundance of the pathway within respective fungal enterotypes relative to that of the others. Asterisks denote the statistical significance of multiple testing corrected Pearson correlation tests (top) and multiple testing corrected Wilcoxon rank-sum tests (bottom): *adjusted p < 0.05, **adjusted p < 0.01, ***adjusted p < 0.001. Stars mark the metabolic pathways involved in carbohydrate degradation. b The relationship between the fungi-contributed pathway PWY-7279 and age. c The relationship between the fungi-contributed pathway PWY-2723 and BMI

Fig. 5

Associations between fungal enterotypes and human diseases. a Enrichment of the fungal enterotypes in human diseases compared to the control group after age was controlled; the odds ratios (OR) and p values of the Fisher’s exact test are shown. AUD: alcohol use disorder; T2D: type 2 diabetes; CDI: clostridium difficile infection; ALHP: alcoholic hepatitis; CD: Crohn’s disease; IBS: irritable bowel syndrome; COVID-19: coronavirus disease 2019; AD: Alzheimer’s disease. b, c Violin plots showing median and quartiles of gut microbiome health index (GMHI) (b) and human DNA contents (HDCs) (c) across fungal enterotypes in the CHGM cohort, where Wilcoxon rank-sum test p values are displayed above the boxplots. d, e Correlations between the HDCs (Y-axis) and the relative abundance of two pathways related to aerobic respiration (X-axis), namely PWY-7279 (d) and PWY-7279 (e). The shaded region denotes the 95% confidence interval of the linear regression. f Mediation linkages among the chronological age, pathway PWY-7279, and HDCs. p_mediation was estimated through a bidirectional mediation analysis with 1000 bootstraps