Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition

Abstract

The microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we discover human- and rodent-associated parabasalid protists, revealing substantial diversity and prevalence in nonindustrialized human populations. Genomic and metabolomic analyses of murine parabasalids from the genus Tritrichomonas revealed species-level differences in excretion of the metabolite succinate, which results in distinct small intestinal immune responses. Metabolic differences between Tritrichomonas species also determine their ecological niche within the microbiota. By manipulating dietary fibers and developing in vitro protist culture, we show that different Tritrichomonas species prefer dietary polysaccharides or mucus glycans. These polysaccharide preferences drive trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.

Keywords: Tritrichomonas; commensal protist; fiber; metabolites; microbiome; microbiota-accessible carbohydrate; mucus; parabasalid; trans-kingdom; tuft cell.

Figure 1.. Identification of parabasalids in mice and humans.

(A) FACS purification of the large T. musculis (Tmu) and small T. casperi (Tc) protists isolated from a mouse cecum. FSC, forward scatter. SSC, side scatter. (B) SEM images of Tmu (left) and Tc (right). UM: Undulating Membrane, RF: Recurrent Flagellum, AF: Anterior Flagella. Scale bars are 2μm. (C) Cladogram of parabasalid protists based on ITS sequences, including mouse and human-associated parabasalids. Protists in bold indicate parabasalids discovered in this study. (D-F) Identification of human-associated parabasalids using mapping-based analysis of metagenomic data. (D) Percentage average nucleotide identity (ANI) of parabasalid metagenomic reads mapping to the corresponding reference protist sequence. Imperfect matches indicate that identified parabasalids are relatives of reference protists and not the same species. First author of the cohort study, country of origin, and population type (“Industrial”, “Transitional”, Hunter-Gatherer (“H-G”)) is indicated. Columns and rows are hierarchically clustered based on Euclidean distance. Cells corresponding to protists not identified in a population are colored grey. (E) Percentage prevalence of subjects in human cohorts with metagenomic reads mapping to reference parabasalids. (F) Prevalence of parabasalids identified in each population type. Asterisks denote protists with imperfect matches to the reference sequence, which are therefore relatives of the indicated species (E, F). (Abbreviations: TZA=Tanzania, NEP=Nepal, MNG=Mongolia, USA=United States of America, MDG=Madagascar, KAZ=Kazakhstan, SWE=Sweden, HMP=Human Microbiome Project).

Figure 2.. T Cell responses to Tmu and Tc differ in the distal SI.

(A) Tuft cell frequency measured by flow cytometry in the distal SI epithelium of uncolonized (Ctrl) mice or those colonized with Tmu or Tc for two weeks. (B) Representative flow cytometry plots of tuft cells. SiglecF marks tuft cells, EpCAM marks epithelial cells. (C) Representative immunofluorescence microscopy images of tuft cell hyperplasia in the distal SI of mice with or without each protist for two weeks. Scale bars are 50μm. (D) Frequency of ILC2s in the lamina propria of the distal SI in Ctrl mice or mice colonized with Tmu or Tc for two weeks. IL13 was measured using the IL13 reporter Smart13 mice (E) Representative flow cytometry plots of ILC2s. (F) Frequency of GATA3⁺ CD4⁺ T cells (Th2 cells) in the lamina propria of the distal SI after three weeks of colonization. (G) Frequency of IFNγ and IL-17 positive CD4⁺ T cells in the distal SI lamina propria after three weeks of Tc colonization, or without protist colonization (Ctrl). Cytokines were stimulated ex vivo and cells were stained for intracellular cytokines. (H) Representative flow cytometry plots of Th1 and Th17 cells in the distal SI with and without Tc. Graphs (A, D, F, G) depict mean. Each symbol represents an individual mouse from two to three pooled experiments (A, D, F, and G). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS, not significant by Student’s t test.

Figure 3.. Th1 and Th17 induction is the shared tritrichomonad-induced immune response.

(A) Frequency (top) and absolute abundance (bottom) of IFNγ and IL-17 positive CD4⁺ T cells in the colonic lamina propria of WT and Caspase1^−/− mice after three weeks of protist colonization, or without protist colonization (Ctrl). (B) Representative flow cytometry plots of the frequency of IFNγ and IL17 positivity in colonic lamina propria CD4⁺ T cells. (C) Frequency and absolute abundance of IFNγ and IL-17 positive CD4⁺ T cells in the distal SI lamina propria of Trpm5^−/− mice after three weeks of Tmu colonization, or without protist colonization (Ctrl). (D) Representative flow cytometry plots of the frequency of IFNγ and IL17 positivity in distal SI lamina propria CD4⁺ T cells from Trpm5^−/− mice with or without Tmu. Cytokines were stimulated ex vivo and cells were stained for intracellular cytokines. Graphs depict mean. Each symbol represents an individual mouse from two (C) or three (A) pooled experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS, not significant by Student’s t test.

Figure 4.. Metabolic differences in tritrichomonads underly divergent immune responses.

(A) Extracellular succinate concentration in the cecal contents of uncolonized mice (Ctrl) or mice colonized with each protist for 3 weeks. (B, C) Genome assemblies of Tmu (B) and Tc (C). Outermost rings show position (in Mbp), middle ring shows genome assembly contigs, inner ring shows GC content in 10kb sliding windows (blue, above genome average; red, below genome average). Total genome size is at the center of each circle. (D) Schematic of metabolic pathways in parabasalid protists. Magenta boxes surround fermentative pathways found in Tc, cyan box surrounds the pathway found in Tmu. (E and F) Gene expression levels (in transcripts per million (TPM)) in the cecum vs. distal SI for Tmu (E) and Tc (F). Transcripts in the lactate and succinate fermentative pathways are labelled if present. (G and H) Intracellular succinate (G) and lactate (H) measurements from purified Tmu and Tc cell lysates. (I) Cladogram of parabasalids. Green shading shows predicted succinate producers. Purple shading shows predicted lactate producers. Asterisks signify metabolic confirmations made in this study. “Human Tritrichomonadida/Trichomonadida” represent previously undescribed human-associated parabasalids detected in Figure 1, but for which sequence information was not obtainable. Phylogenetic orders are labelled on the right. Graphs (A, G, H) depict mean with SD. *p<0.05, **p<0.01, NS, not significant by Student’s t test.

Figure 5.. Tmu and Tc occupy different nutritional niches within the microbiota.

(A) Abundance of Tmu and (B) Tc in the stool of mice fed diets with defined fiber compositions for two weeks or fed standard chow (complex) as a control. Fibers in the defined diets are limited to inulin (I), cellulose (C), or no fiber (None). (C) 16S rRNA amplicon sequencing in the colon of uncolonized (Ctrl) mice or mice colonized with Tmu or Tc for three weeks. The 15 most abundant taxa are shown, and data is the average abundance from five mice per group. (D) Relative abundance of bacteria depleted by Tmu and Tc colonization in each region of the intestine, from 16S Sequencing. (E) Representative microscopy showing localization of Tmu and Tc in the colons of mice fed complex chow. Scale bars are 100μm. (F) Quantification of protist localization by microscopy. Higher mucus/lumen protist signal indicates tighter localization to the colonic mucus layer. (G) Number of CAZymes encoded by protists and bacterial competitors with predicted activity against host and plant glycans. (H) Abundance (in TPM) of putative mucus-utilization genes in Tc and Tmu from transcriptomics data. CAZymes were identified by dbCAN2. For putative mucolytic CAZymes the glycoside hydrolase family (GH) is indicated in brackets. Graphs (A, B, D, F) depict mean, error bars depict SD in (D). Each symbol represents an individual mouse from one to three pooled experiments (A). **p<0.01, ***p<0.001, ****p<0.0001, NS, not significant by Mann-Whitney test (A, B) or Student’s t test (F).

Figure 6.. Dietary MAC deprivation causes Tmu to switch to a mucolytic metabolism.

(A) Representative images of Tmu in the colons of mice fed complex chow (left) and defined chow with cellulose (def-C) chow (right) for two weeks. Scale bars are 100μm. Inset shows mucus-associated Tmu during fiber starvation. (B) Quantification of microscopy on Tmu localization to the mucus layer when mice are fed complex chow or def-C chow. Higher mucus/lumen protist signal indicates tighter localization to the colonic mucus layer. (C) Growth curves of Tmu cultured in vitro, using the PBF medium that we developed, with maltose (cyan) or mucus (green) added as carbon sources, or with no defined carbon source added (grey). (D) Differential gene expression from Tmu grown in vitro with either maltose or mucus as a carbon source. Green data points indicate putative mucus-utilization genes with increased expression when grown in the presence of mucus. GH families for putative mucolytic CAZymes are labelled. Dashed lines delineate the fold change cutoff (1.5-fold). (E) Tmu abundance in the stool of mice fed def-CI or def-C chow for two weeks, with or without an antibiotic cocktail (VNA). Antibiotics used are Vancomycin, V; Neomycin, N; Ampicillin, A. (F) Tmu abundance in the cecal contents of mice fed cellulose chow for two weeks with different antibiotic treatments. (G) 16S rRNA sequencing of stool from Tmu-colonized mice fed def-C chow for two weeks with different antibiotic treatments. Each column represents an individual mouse. (H) Relative abundance of known mucolytic bacteria in mice from each group in (G). LD, limit of detection. Graphs (B, C, E, F, H) are plotted as mean, error bars depict SD in (C). Each symbol represents an individual mouse from one to three pooled experiments (B, E, and F). **p<0.01, ****p<0.0001, NS, not significant by Mann-Whitney test (E, F) or Student’s t test (B).

Figure 7.. Diet-protist interactions shape the host immune response.

(A) In vitro growth curve of Tmu cultured in PBF medium with maltose or inulin added as carbon sources, or with no defined carbon source added. (B) Representative images of Tmu in the colons of mice fed complex chow or defined chow with cellulose and inulin (def-CI) for three weeks. Scale bars are 50μm. (C) Quantification of microscopy on Tmu localization to the colonic mucus layer when mice are fed complex chow or def-CI chow. Higher mucus/lumen protist signal indicates tighter localization to the colonic mucus layer. (D) Relative abundance of Bacteroidetes bacteria in the stool of mice colonized by Tmu or Tc, with the fermentable fiber inulin or the non-fermentable fiber cellulose as the only fiber sources in the diet. (E) Maximum Tmu culture density when grown in vitro with different dietary MACs added as carbon sources to PBF medium. All statistical comparisons are relative to culture with no defined carbon source added. (F) Differential expression of Tmu transcripts grown in vitro with either maltose or arabinoxylan as a carbon source. Cyan data points represent upregulated CAZymes with potential activity towards arabinoxylan. Dashed lines delineate the fold change cutoff (1.5-fold). (G and H) Epithelial tuft cell (G) and lamina propria Th2 (H) frequency in the distal SI of mice colonized with Tmu or no protist (Ctrl) and fed complex or def-CI chow for 3 weeks. (I) Tmu abundance in the distal SI of mice fed complex chow and def-CI chow for 3 weeks. Data is plotted as mean with SD (A and E). Each symbol represents an individual mouse from two or three pooled experiments (C, D, G, H, and I). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS, not significant by Mann-Whitney test (D, I) or Student’s t test (C, E, G, H).