Commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota

Abstract Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus and intestinal bacteria in healthy and B-cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with Tritrichomonas musculus functional changes, which were accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single-cell transcriptomics identified distinct Tritrichomonas musculus life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable data sets to drive future mechanistic studies.

post colonization.Protists were purified from caecal contents and immediately transferred on ice to the Princess Margaret Genomics Centre (Toronto, Canada) for STAMP library preparation using Drop-seq technology, and sequenced on a NextSeq 500 System (Illumina, San Diego, CA, USA) 19 .Reads were processed using Drop-seq Tools v.1.13and aligned to the protist genome assembly using STAR v. 2.5.3a 20,21.Three thousand protists per mouse (minimum 200 genes, 500 transcripts) were analyzed using Seurat v4 22,23 .Cells were grouped using graph-based clustering (0.8 resolution, 18 principal components) and visualized via UMAP 24 .Differentially expressed (DE) genes were identified using the FindAllMarkers function, and functional enrichments were determined based on overrepresentation of pathway enzymes as defined by KEGG using the hypergeometric test or GO terms using the topGO package and the Fisher's Exact test 25,26 .Enrichments of custom-defined gene sets (meiosis, G1/S, and G2 phase genes) were scored with the AddModuleScore function and evaluated using two-sided Wilcoxon rank-sum tests.Benjamini-Hochberg correction was applied for multiple testing 27 .Heatmaps were generated using pheatmap 1.0.12 and Ward.D2 clustering 28 .qPCR DNA primers used for quantitative PCR of bacterial taxa 29

RNAScope
RNAScope was performed as per the RNAScope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Newark, CA, USA) protocol.Approximately 0.5 cm caecum sections were excised from Tmucolonized mice, placed in 10% neutral buffered formalin, and fixed overnight at room temperature (RT) with gentle agitation.The following day, samples were washed with PBS, placed in 70% ethanol, embedded in paraffin, and sliced to 7 μm sections at the Toronto Centre for Phenogenomics.Paraffin sections were baked at 40°C for 30 min in a HybEZ Oven (Advanced Cell Diagnostics, Newark, CA, USA), and treated with hydrogen peroxide at RT for 10 min.Antigen target retrieval was conducted at 99°C under the 15 min standard procedure.A barrier was created around sections using an ImmEdge pen (Vector Laboratories, Burlingame, CA, USA) and allowed to dry for 15 min.Samples were treated with protease at 40°C for 30 min and stored in saline sodium citrate solution overnight (175.3 g NaCl, 88.2 g sodium citrate, 800 mL ddH2O, pH 7).TSA Plus Fluorophores Fluorescein and Cyanine 3 were hybridized against protist probes TMU_00005724 and TMU_00016742 respectively, and samples were visualized using a Zeiss AXIO Observer microscope (Carl Zeiss AG, Jena, Germany).Resin blocks were sectioned to 80 nm thickness with a Reichert Ultracut E microtome (Leica, Wetzlar, Germany), collected on 300 mesh copper grids (EMS), and counter stained for 10 min using saturated 5% uranyl acetate followed by Reynold's lead citrate (EMS).Prepared grids were placed on a filter paper mat in labelled Petri dishes and stored in a desiccator until imaging.The sections were imaged using a Talos L120C transmission electron microscope (Thermo Scientific, Waltham, MA, USA) equipped with a BM-Ceta scientific CMOS camera at an accelerating voltage of 120 KV.  [Eubacterium] ventriosum group Muribac.

1 .
Microbial interaction networks depicting interbacterial and Tmu-bacterial correlations over time.Nodes represent bacteria or Tmu, as indicated, and were clustered using the Markov Cluster Algorithm.Node colours represent the abundance pattern identified in Fig.1g, and border colours the taxonomic phylum, as detailed in the legend.Edges are positive (red) or negative (blue) correlations based on Kendall τ coefficients (strength of correlation ≥ 0.7).Bacteria directly correlated with Tmu are named and linked with thick lines.Muribac.= Muribaculaceae.

Lachnospiraceae Supplementary Figure 2 .Supplementary Figure 3 . 5 .Supplementary Figure 6 . 7 .
Caecal metatranscriptomics of Tmu-colonized and naïve (control) WT and muMt -/- mice.a, Breakdown of caecal RNA reads from filtering and annotation steps.Columns represent samples from individual mice.Outset graph to the right shows percentages of putative mRNA reads mapped to the Tmu genome assembly.b, Taxonomic classification of putative bacterial transcripts.putative bacterial mRNA (unannotated) putative bacterial mRNA (annotated) Upregulation of bacterial metabolism in response to protist colonization.Depicted are glycolysis/gluconeogenesis (ec00010), tricarboxylic acid (TCA) cycle (ec00020) and the pentose phosphate (PP) (ec00030) pathways in gut microbiota after 2 or 28 days of infection in WT or B cell-deficient (muMt -/-) hosts.Genes significantly up-and downregulated (p <0.05 in DESeq2 analysis) are indicated with blue and red borders, respectively.Sizes of nodes represent log2 fold-changes between Tmu-colonized and uninfected control mice (n=4 per group).Pie charts depict the phylogenetic source of the gene expression as follows: yellows represent Proteobacteria or Desulfobacterota; orange is Campilobacterota (Helicobacter); shades of green are Bacteroidota (dominated by Bacteroides and Parabacteroides); pinks and purples are Firmicutes (dominated by Lachnospiraceae and Clostridium); black represents unclassified bacteria.Putative virulence-related Tmu genes differentially expressed over the course of colonization.a, Lectin and b, cysteine proteases differentially expressed at 28 compared to 2 days post colonization in WT or muMt -/-mice as indicated.c, BspA-like genes with significant changes in expression over colonization time and/or which differ between host genotypes.d, Expression of genes with predicted activity in the arginine dihyrdolase pathway: arginine deiminase (ADI), ornithine carbamoyltransferase (OCT), and carbamate kinase (CK).Di fferences in gene expression were tested using DESeq2.*p <0.05, ** <0.01, *** <0.001, ns non-significant Expression of cell cycle marker genes.a, Dotplot and b, heatmap depicting scaled read counts of genes known to be expressed during G1/S and G2 phases across each Tmu cluster.Colour blocks in b (left) indicate assigned gene function.Counts are separated by the host mouse.c, Expression of meiosis-specific genes.WGA-staining of Tmu cells freshly isolated from mouse caeca or colons, or in vitro cultured for 1 to 3 days.a, Representative contour plots from FACS analysis of WGA-FITC stained protists.Events are gated on live single protists.n=3 animals or culture plates per group from four independent experiments.b, Cytospins of WGA-FITC (green) and DAPI (blue) stained protists.Representative images are shown at 63x magnification.Scale bars,

Table 2 .
Tmu single cell RNA sequencing statistics.
a Read mapping percentages were calculated as a proportion of quality filtered reads.bOne cell removed due to low gene and transcript detection.