A unique symbiosome in an anaerobic single-celled eukaryote

Abstract

Symbiotic relationships between eukaryotes and prokaryotes played pivotal roles in the evolution of life and drove the emergence of specialized symbiotic structures in animals, plants and fungi. The host-evolved symbiotic structures of microbial eukaryotes - the vast majority of such hosts in nature - remain largely unstudied. Here we describe highly structured symbiosomes within three free-living anaerobic protists (Anaeramoeba spp.). We dissect this symbiosis using complete genome sequencing and transcriptomics of host and symbiont cells coupled with fluorescence in situ hybridization, and 3D reconstruction using focused-ion-beam scanning electron microscopy. The emergence of the symbiosome is underpinned by expansion of gene families encoding regulators of membrane trafficking and phagosomal maturation and extensive bacteria-to-eukaryote lateral transfer. The symbionts reside deep within a symbiosomal membrane network that enables metabolic syntrophy by precisely positioning sulfate-reducing bacteria alongside host hydrogenosomes. Importantly, the symbionts maintain connections to the Anaeramoeba plasma membrane, blurring traditional boundaries between ecto- and endosymbiosis.

Fig. 1. Anaeramoeba and symbionts.

a Scanning electron micrograph (SEM) of an Anaeramoeba flamelloides BUSSELTON2 amoebae showing a hyaline front and posterior trailing projections. Scale bar 5 µm. b Immunolocalization of alpha-tubulin (TAT1 antibody, 1:200) in A. flamelloides BUSSELTON2 cells using laser scanning confocal microscopy. Host nuclei and symbiont DNA were stained using DAPI (top panel) and the acentriolar centrosome and radiating microtubules by alpha-tubulin TAT1 antibody (middle panel) with overlay (bottom panel). Scale bar 5 µm. c TEM of chemically fixed A. flamelloides BUSSELTON2. Scale bar 5 µm. d TEM of chemically fixed A. flamelloides BUSSELTON2. Scale bar 2 µm. e TEM of cryo-fixed A. flamelloides BUSSELTON2. White arrow shows acentriolar centrosome. Scale bar 2 µm. f Transmission electron micrograph (TEM) of A. ignava BMAN showing vesicle-bound symbionts (S) and host hydrogenosomes (H) in close proximity. The boxed inlay shows narrow openings (red arrow) connecting outside media to the vesicles housing the symbionts. Scale bar 1 µm. Nucleus (N), Food-vacuole (fv). The experiment in (b) shows representative cells from three independent replicate immunostainings.

Fig. 2. Anaeramoeba symbionts are housed in a membrane network with connections to the plasma membrane.

a–c FIB-SEM slice of A. flamelloides BUSSELTON2, showing (b) segmented regions of interest (symbiont–blue, hydrogenosome–red, symbiosome membrane–gold, nucleus–purple, granules–aubergine, other prokaryotes– blue green, plasma membrane–yellow). c overlay of (a, b). d–f Rendered volume of FIB-SEM slices showing segmented regions of interest. d Symbiont, symbiosome membrane, nucleus and plasma membrane. e Hydrogenosomes, nucleus and plasma membrane. f Overlay of all regions of interest in (d, e), and microtubules (MTs) (symbiont–blue, hydrogenosome–red, symbiosome membrane–gold, nucleus–purple, plasma membrane– yellow, MTs–pink). g A clipped 3D rendering of A. flamelloides BUSSELTON2 symbionts (blue), hydrogenosomes (red), symbiosome membrane (gold) and nucleus (purple) showing the internal structure of the symbiosome. h–i Two different views of 3D surface rendering of the components of the symbiosome connected to the plasma membrane (light blue). Plasma membrane (yellow) and nucleus (purple). Symbiosome connections to the plasma membrane are indicated by black arrows. Scale bar (a–c), 1 µm.

Fig. 3. Anaeramoeba symbionts are closely related to Desulfobacter and acquired in separate events.

A. ignava BMAN stained with (a), DAPI and hybridized with (b), probe DSBA355-BMN-488, (c), probe Delta495a-Atto 550 and (d), probe EUB338a-Atto 633. A. flamelloides BUSSELTON2 stained with (e), DAPI and hybridized with (f), probe BUSS/SCH-BMN-488, (g), probe Delta495a-Atto 550 and (h), probe EUB338a-Atto 633. A. flamelloides SCHOONER1 stained with (i), DAPI and hybridized with (j), probe BUSS/SCH-BMN-488, (k), probe Delta495a-Atto 550 and (l), probe EUB338a-Atto 633. m The phylogenomic analysis was based on 36 taxa, 108 proteins, and 25,918 sites with IQTree v2.2.0.3 (LG + C60 + F + G model of evolution). Bipartition support values are derived from 100 non-parametric bootstraps under the PMSF model. Scale bar indicates inferred number of substitutions per site. Tree files and alignments are available at FigShare: 10.6084/m9.figshare.20375619. Scale bar (a–l), 1 µm. The experiments in (a–d), and (e–l), show representative images from duplicate and triplicate hybridizations respectively.

Fig. 4. Anaeramoeba symbionts are metabolically poised for syntrophic interaction with hydrogenosomes.

Suggested syntrophic interactions between Anaeramoeba hydrogenosomes (blue) and symbionts (pink/salmon) based on metabolic reconstruction from transcriptomic and genomic evidence. The ATP-producing hydrogenosomes generate H₂, acetate, and propionate as end-products (in bold). Based on metatranscriptomic data, the symbionts use the hydrogenosome products by prominently expressing the dissimilatory sulfate reduction (DSR), methylmalonyl-CoA, and the Wood-Ljungdahl pathways. The symbionts are in deep membrane-pits with a connection to the cell surrounding that gives ready access to sulfate (gold). HynAB periplasmic [NiFe] hydrogenase, HysAB [NiFeSe] hydrogenase, Qmo QmoABC complex, Tmc TmcABCD complex, Hdr Heterodisulfide reductase, Hme DsrMKJOP complex, Nqr NADH:ubiquinone oxidoreductase, Rnf Rnf complex, Atp ATP synthase, APS adenosine 5'-phosphosulfate, CoA Coenzyme A, NAD Nicotinamide adenine dinucleotide, NADPH Nicotinamide adenine dinucleotide phosphate, Fd_red/ox Ferredoxin reduced/oxidized, ATP adenosine triphosphate.

Fig. 5. Taxonomic range and functional categories of LGT donors in Anaeramoeba.

a Taxonomy of LGT donors for genes inferred to have been acquired in the common ancestor of Anaeramoeba and separately in A. flamelloides and A. ignava. The numbers on the branches are the inferred number of LGTs on each position of the tree. b Breakdown of LGTs from proteobacterial donors. c Functional categories of genes inferred to have been acquired in the Anaeramoeba common ancestor, in A. flamelloides and A. ignava. Color coding is the same as for part (b). d Maximum-likelihood phylogeny of FAD-dependent oxidoreductases. e Maximum-likelihood phylogeny of vitamin B12-dependent methionine synthase MetH. The trees shown in (d) and (e) were produced by our LGT-detection pipeline (see text). f Maximum-likelihood phylogeny of oxidosqualene cyclase (OSC), squalene-hopene cyclase (SHC), and squalene-tetrahymanol cyclase (STC). The tree shown stems from analyses based on previously published datasets^,. Sequences were aligned with MAFFT, sites were selected using BMGE, and the phylogeny inferred using IQTree model C20 + G4 with 1000 ultrafast bootstraps. Anaeramoeba sequences are in red, eukaryotic sequences in blue, and prokaryotic sequences in bright green. Scale bars indicate the inferred number of amino acid substitutions per site. Abbreviations: INFORMATION STORAGE AND PROCESSING: [J] Translation, ribosomal structure and biogenesis; [K] Transcription; [L] Replication, recombination and repair; CELLULAR PROCESSES AND SIGNALING: [D] Cell cycle control, cell division, chromosome partitioning; [V] Defense mechanisms; [T] Signal transduction mechanisms; [M] Cell wall/membrane/envelope biogenesis; [N] Cell motility; [Z] Cytoskeleton; [W] Extracellular structures; [U] Intracellular trafficking, secretion, and vesicular transport; [O] Posttranslational modification, protein turnover, chaperones. METABOLISM: [C] Energy production and conversion; [G] Carbohydrate transport and metabolism; [E] Amino acid transport and metabolism; [F] Nucleotide transport and metabolism; [H] Coenzyme transport and metabolism; [I] Lipid transport and metabolism; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport and catabolism. POORLY CHARACTERIZED: [R] General function prediction only; [X] Mobilome: prophages, transposons; [S] Function unknown.