Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system

Johnson, Matthew D.; Shepherd, Doulin C.; Sakai, Hiroyuki D.; Mudaliyar, Manasi; Pandurangan, Arun Prasad; Short, Francesca L.; Veith, Paul D.; Scott, Nichollas E.; Kurosawa, Norio; Ghosal, Debnath

doi:10.1038/s41467-024-51159-2

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Published: 16 August 2024

Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system

Nature Communications volume 15, Article number: 7066 (2024) Cite this article

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Abstract

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.

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Introduction

DPANN, an acronym of the names of the first included 5 phyla (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota)¹, is a superphylum containing >10 phylum-level lineages, most of which remain uncultivated. The common features of DPANN lineages are small cell and genome sizes with very limited metabolic capabilities². Although rare free-living DPANN lineages exist, they mostly adopt an obligate symbiotic lifestyle dependent on other microbial community members^3,4. DPANN accounts for approximately half of all the archaeal diversity^2,5, and is detected across the entire biosphere^{3,4,5,6,7,8,9,10,11,12,13,14}, including within the human microbiome⁶. However, their biological functions, cell biology and molecular basis of symbiosis are still poorly characterized since only three representative phyla have ever been co-cultured^{7,8,9,10,11,12,13}.

One of the DPANN lineages, the phylum Microcaldota^14,15 (formerly “Ca. Micrarchaeota”), originally called ARMAN¹⁶, has been found in various natural environments (e.g., acid mine drainages (AMD), hot springs, soil, peat, hypersaline mats, and freshwater^17,18). Since the first discovery of ARMAN in AMD¹⁶, its host has been generally suggested to be a member of the order Thermoplasmatales (the phylum Euryarchaeota)^19,20. However, we have recently established a co-culture system composed of a Micrarchaeon Microcaldus variisymbioticus ARM-1 (hereon referred to as ARM-1) and its host Metallosphaera javensis AS-7 (hereon referred as AS-7) belonging to the order Sulfolobales (the phylum Crenarchaeota)¹⁴. Notably, most Thermoplasmatales spp. lack a surface layer (S-layer)²¹, while Sulfolobales spp. have a defined S-layer²². This difference in the cell envelope, in combination with other unknown factors, could contribute to host specificity of the Microcaldota.

Previous studies suggested that DPANN-host interaction could involve membrane fusion and direct cytoplasmic connection (or, “cytoplasmic bridge”) between the host and the symbiont, facilitating the exchange of nutrients and even enzymes^{11,19,20,23,24,25,26}. The molecular and structural basis of host-DPANN attachment, “cytoplasmic bridge” formation, and host-DPANN symbiosis remain poorly understood. Intriguingly, a few DPANNs were recently reported to engage with their hosts without the apparent presence of a cytoplasmic bridge^14,27,28. How intercellular communication is facilitated in the absence of a direct cytoplasmic connection is unclear.

Recent advances in electron cryo-tomography (cryo-ET) and associated methods have enabled the investigation of cellular ultrastructures and large macromolecular machines in situ in their near-native state at nanometer resolution^29,30,31. Here, we use cryo-ET and complementary approaches, including proteomics and genome analysis to investigate the interaction between Microcaldota ARM-1 and its host AS-7 at molecular resolution. Our in situ structural analyses reveal a proteinaceous nanotube structure that bridges the cytoplasms of AS-7 and ARM-1 and might act as a conduit for molecular exchange. We resolve the structural details of the host and DPANN S-layers, and the bridging tube connecting the two cell types. Finally, our proteomic analysis revealed host response upon DPANN attachment and provided insights into the molecular basis of host-DPANN symbiosis.

Results

Cryo-ET uncovers nanometer-resolution details of host-DPANN interaction

We utilized cryo-ET to visualize the interactions between ARM-1 and AS-7 in their near-native state, at nanometer resolution. ARM-1 was co-cultured with host AS-7 using methods previously described¹⁴ and the growth of the two cultures was monitored by qPCR using 16 S-specific primers (Supplementary Fig. 1A). Association between ARM-1 and AS-7 was initially assessed by scanning electron microscopy (SEM) and subsequently imaged by cryo-ET (Supplementary Fig. 1B–D). Our cryo-tomograms showed multiple ARM-1 Microcaldota interacting tightly with each AS-7 cell (Fig. 1A, B, Supplementary Movies 1 and 2). The different cell types (host and DPANN) are distinguishable by the appearance of their S-layers and overall sizes; the AS-7 host cell has a characteristic corrugated S-layer and measures 1.5–2 µm in diameter, while ARM-1 measures 0.3–0.5 µm in diameter (Fig. 1A, B and Supplementary Fig. 1B–D). Density profile analysis of the ARM-1/AS-7 envelopes revealed that the distance from ARM-1 S-layer to AS-7 S-layer was between 11 and 16 nm (Supplementary Fig. 2). Close examination of AS-7 cells showed proteinaceous “tube-like” structures embedded just beneath the S-layer (Fig. 1C, E, yellow arrows). Intriguingly, we also observed many of these nanotubes extending between the two cell types bridging their cytoplasms (Fig. 1A, C). Nanotubes embedded within the AS-7 S-layer are likely the primed state that, upon some stimulation, extends to form host-DPANN contact. The number of extended nanotubes varied between different ARM-1 and AS-7 interactions with an average of 4.4 nanotubes per symbiont/host contact (Supplementary Fig. 3A, B). In some of our tomograms, we observed more than 10 nanotubes connecting each ARM-1 to its host, whereas in a few others, there were none (Supplementary Fig. 3A, B).

**Fig. 1: Molecular interactions of the ARM-1—AS-7 co-culture.**

Top views of our reconstructed tomograms revealed that the AS-7 S-layer forms a uniform lattice with pores of P6 symmetry. The hexagonal arrangements often had a circular structure at the center (Fig. 1D). Careful investigation of our tomograms from different axes and measurement of dimensions confirmed that these circular structures are top views of the nanotubes embedded within the AS-7 S-layer (Fig. 1D, E). Interestingly, the nanotubes were structurally aligned to the S-layer pores, suggesting that the AS-7 S-layer likely forms a guiding scaffold for the tube assembly and extension. The frequency of nanotubes varied between different tomograms and had a positive correlation with the presence of the symbiont.

In many of our tomograms, we observed several thin filament-like “antennae” densities protruding out from the S-layer of AS-7 cells into the extracellular environment (Fig. 1E, white arrows). We were unable to resolve if the antennae traversed the entire cell envelope. The tip of the antennae harbored a bulb-like density reminiscence of bacteriophage tail fibers³². The antennae might play a role in establishing the initial contact before host-DPANN cytoplasms are connected by nanotubes. These results show the nature of ARM-1 and AS-7 interaction in molecular detail and reveal previously uncharacterized protein nanotube and putative receptor structures that mediate host-DPANN interaction.

In situ structural analyses of the AS-7 S-layer show protein nanotubes originate from the host

Tomographic slices parallel and perpendicular to the AS-7 S-layer revealed that the S-layer formed a 2D regular hexagonal array, which is also confirmed by the power spectrum analysis (Supplementary Fig. 4A–D). To obtain a detailed understanding of the surface organization of AS-7 and resolve the in situ structure of the nanotube in its primed state, we performed subtomogram averaging (STA) of the AS-7 S-layer with and without the nanotube.

For the AS-7 S-layer, we first manually picked 200 particles to produce a de novo model in EMAN2^33,34. This model was then used as a reference for template matching to pick 10,711 particles from 72 tomograms. STA of the AS-7 S-layer lattice resulted in a ~1 nm resolution structure of the S-layer, where S-layer protein (SlaA and SlaB) domains were distinctly resolved (Fig. 2A–C, Supplementary Fig. 4A, E). This allowed us to generate AlphaFold models of SlaA and SlaB and fit those in our subtomogram average map, revealing overall orientation and organization of SlaA and SlaB within the S-layer lattice (Supplementary Fig. 4F–I, Supplementary Movie 3). The local resolution of our structure was 8–16 Å as estimated by ResMap³⁵ (Global resolution of 14.8 Å as calculated by FSC) (Supplementary Fig. 5). Our structure showed that the S-layer formed a regular array of dome-like structures at a center-to-center distance of ~21 nm (Fig. 2C). The dome-like structure was rotationally 6-fold symmetric with a small pore in the center, which had a diameter of 3.5 nm (Fig. 2A, C).

**Fig. 2: In situ structure of the AS-7 S-layer and primed nanotube.**

To determine the structure of the embedded/primed protein nanotube, we manually picked 797 side and top views of nanotubes embedded within the S-layer and generated an STA (Fig. 2D–G). The local resolution of the protein nanotube structure varied between 10 and 20 Å, as calculated by ResMap (Global resolution of 27.3 Å as calculated by FSC) (Supplementary Fig. 6). The resulting average revealed that the envelope-spanning nanotube was assembled within the S-layer scaffold and extended from the dome to the cytoplasmic membrane (Fig. 2E–G). The nanotube was structurally aligned with the pore of the S-layer, suggesting that the S-layer pore could act as conduit for nanotube extension during host interaction (Fig. 2G). The base of the nanotube, near the cytoplasmic membrane, was narrower suggesting there might be a gating mechanism. The top and vertical cross-section views of the average best illustrated the architecture of the membrane-spanning complex, but the overall organization was better viewed in the segmented volume (Fig. 2E–G, Supplementary Movie 4). The segmented volume highlighted 6 necklace-with-pendant-like densities around the tube, extending down from the S-layer (Fig. 2E), providing a further framework for the nanotube structure. The length of the nanotube from the dome to the cytoplasmic membrane was 28.6 nm and the inner and outer diameters were 4.4 and 6.7 nm, respectively, suggesting a wide enough lumen for metabolite and unfolded protein transport (Fig. 2G). Given the AS-7 S-layer pore diameter is 3.5 nm, the nanotube extension cannot occur without a large conformational change within the S-layer or a symmetry break. Since nanotubes were also present in AS-7 pure culture (Supplementary Movie 5) and were integrated within the AS-7 S-layer, the connecting tubes originate in the AS-7 host cells and not the ARM-1 DPANN.

In situ structure of the AS-7 extended nanotube

To understand the structural basis of host-DPANN interaction, we attempted to generate an STA of the extended tube connecting cells. Our initial attempts to average the extended tube using EMAN2 were unsuccessful due to the considerable range of angles that the tube protruded from the AS-7 envelope (Supplementary Fig. 7). Therefore, we adopted a guided approach where relative orientations for each particle within cryo-tomograms were provided as a priory information for further focused alignment and refinement. In our initial average, part of the tube entering the ARM-1 envelope resolved well but densities around the AS-7 S-layer were lacking. Therefore, we used masks to perform focused alignment and averaging of the tube near the ARM-1 envelope separately from that of the AS-7 envelope. By combing the two averages, we generated a composite STA of the intercellular nanotube bridging the DPANN and host (Fig. 3A, B). Our structure showed that the nanotube originates in the AS-7 cytoplasmic membrane and traverses through the AS-7 and ARM-1 S-layers, which we interpret connects their cytoplasms (Fig. 3A, B) though the limited resolution will require further confirmation. We also cannot unambiguously exclude the possibility these observations could be due artefacts caused by sample preparation and will require further investigation. The extended nanotubes are 68 nm long, with an outer and lumen diameter of 6.7 and 4.4 nm, respectively (Fig. 3B). Intriguingly, we observed a barrel-like structure in the ARM-1 envelope seemingly acting as a docking platform for the in-coming tube. The barrel-like structure is reminiscent of the type-II secretion system secretin complex but with a much larger diameter of 21 nm (Fig. 3B)³⁶. Because the tubes emanated at diverse angles from the AS-7 envelope, despite a guided approach and focused alignment, the AS-7 S-layer resolved poorly in our composite average. (Fig. 3B, Supplementary Fig. 7).

**Fig. 3: Architecture of the DPANN (*Microcaldus variisymbioticus* ARM-1) S-layer and extended intercellular nanotube.**

The ARM-1 outer membrane is comprised of an S-layer

No structural elucidation of the S-layer has been achieved from any of the DPANN superphylum members to date. Our attempts to generate an STA of the ARM-1 S-layer using multiple different software were unsuccessful. This could be because of the highly bent nature of the ARM-1 S-layer (due to small size and curvature) and/or imperfect lattice arrangement. Unlike the host AS-7 S-layer, no corrugated dome-like arrangement was observed in the ARM-1 envelope and only one ortholog for SlaA was found in the ARM-1 genome, suggesting a different S-layer organization. Side views of the ARM-1 envelope showed crescent-shaped structures, extending downwards, with three discrete densities extending from the outermost layer towards the cytoplasmic membrane (Fig. 3C, inset). The distribution and orientation of these densities were visibly irregular, suggesting imperfect lattice arrangement. In a few tomograms of partially lysed ARM-1 cells, S-layers were found partly detached from the main cell body (Fig. 3D). Recent studies have used methods to artificially shed S-layers from cells using detergents in order to obtain native structures of S-layers³⁷. We used this opportunity to generate an average of the ARM-1 S-layer top view (Fig. 3D, inset). Our average showed that the ARM-1 S-layer constitutes an ordered 2D S-layer lattice of sixfold symmetry with a pore in the center. The center-to-center distance between neighboring hexametric arrangements was 18.5 nm.

Comparative proteomics of the host and co-culture reveals molecular players involved in the symbiotic relationship

Our cryo-ET analysis revealed that the host archaeal cell AS-7 interacts with the ARM-1 symbiont through intercellular nanotubes, and that these tubes originate and extend from the AS-7 host. To gain insights into host response to ARM-1 interaction and identify plausible resources being exchanged between the host and the symbiont, we performed comparative proteomics of pure host and host-DPANN co-cultures. Using Label-free quantification (LFQ) based proteomics a total of 698 ARM-1 proteins and 1810 AS-7 proteins were identified with the presence of ARM-1, resulting in widespread alterations within the AS-7 proteome (Fig. 4A, B). Of the 698 proteins identified from ARM-1, 637 were robustly identified across all samples with sequence analysis demonstrating much of the observed proteome lacks identifiable PFAM domains or similarities to known proteins based on GO and KEGG assignments (Supplementary Fig. 8A). Despite this, analysis of the relative abundance of the observable proteome based on iBAQ analysis³⁸ (Supplementary Fig. 8B) supports that the observable proteome spans nearly 5 orders of magnitude and reveals proteins associated with ATP synthase are highly abundant, consistent with the active growth of ARM-1. Within the observable AS-7 proteome, 134 proteins were significantly increased in abundance while 373 were decreased upon ARM-1 interaction (Fig. 4A–C). Overlaying these observed proteomic changes on the genome revealed multiple loci to display congruent changes within neighboring gene products (Fig. 4D–F, Supplementary Data 1). For example, the addition of ARM-1 resulted in an increase in multiple gene products of BCS91585…1-BCS91596.1 and BCS93590.1…BCS93624.1, with these proteins associated with CO₂ fixation, and the TCA cycle; BCS92975.1…BCS92983.1, encodes proteins that are predicted to be involved in inorganic ion transport and metabolism (Fig. 4E, cluster i); and BCS93537.1…BCS93550.1 involved in aromatic metabolism, tyrosine metabolism, and branched-chain amino acid metabolism (Fig. 4F, cluster ii). Comparative genomics analysis of these impacted AS-7 proteins supports that cluster (ii) is found exclusively in AS-7 suggesting a role for this locus for the AS-7 and ARM-1 interaction (Supplementary Fig. 9, Supplementary Data 2).

**Fig. 4: Proteome analysis of *Microcaldus variisymbioticus* ARM-1 and *M. javensis* AS-7.**

COG analysis of the 373 significantly decreased proteins in the AS-7 reveals that most differentially downregulated proteins appear involved in translation, ribosomal structure, and biogenesis (COG:J), transcription (COG:K), and replication, recombination, and repair (COG:L) (Fig. 4G). Specifically, a four gene operon (BCS92057.. BCS92060) encoding proteins for the exosome complex, which is responsible for RNA degradation; a four gene operon (BCS91961..BCS91964), containing three hypothetical proteins that have predicted structures similar to SNARE proteins that are involved in vesicle formation and trafficking; a six gene operon (BCS94267..BCS94277) containing Alba DNA binding proteins and DNA topoisomerases. Collectively, these results indicated that the AS-7 cell is reducing its proliferation rate in response to ARM-1 interaction.

Filamentous structures, protein cages, and large membrane tubes between ARM-1 cells

Interestingly, in some of our tomograms, ARM-1 cells had stacks of filament-like structures close to the membrane, resembling filamentous phages³⁹ (Supplementary Fig. 10A). We also noticed, large protein-cage-like structures that were often more than 100 nm long and 50–70 nm wide assuming barrel or trapezoid-like shapes (Supplementary Fig. 10B, Supplementary Movie 2). These assemblies are similar to those previously reported⁴⁰, despite the improved resolution presented here, their function remains unknown. In addition, we observed thin filaments protruding out of ARM cells that could represent archaeal pili (Supplementary Fig. 10C). Finally, in a few of our tomograms, we saw two ARM-1 cells were connected by wide membrane tubes (Supplementary Fig. 10D–F, Supplementary Movie 6). Such nanotubes/nanopods have been previously described in Crenarchaeota and Euryarchaeota but their exact function remains unclear⁴¹.

Discussion

Here, using complementary approaches, including in situ structural biology, proteomics, and genomic analysis, we investigated the structural and molecular basis of the episymbiotic relationship between Microcaldus variisymbioticus ARM-1, a DPANN archaea, and its archaeal host M. javensis AS-7. Our results characterize the host-symbiont interactions at molecular resolution revealing an antenna-like structure on the surface of the host, and an intercellular nanotube structure, bridging the host and DPANN cytoplasms. The antenna structure could mediate symbiont recognition and initial contact formation, leading to the biogenesis of the nanotube apparatus. Previous low-resolution studies have shown that DPANNs can form different types of interactions with their hosts including: cytoplasmic bridges¹⁹, parasite-like invasion^11,28, and pili-like connections^19,42. Here, we discovered a nanotube structure that may bridge the host and symbiont, promoting intercellular exchange. Our nanotube structures have a resemblance to previously described connections between Thermoplasmatales and associated DPANN (ARMAN). Due to limited resolution, these connections were referred to as “filamentous connections”^19,42. Thermoplasmatales archaea are distantly related to Metallosphaera, indicating that these nanotubes maybe conserved across different archaea. Our cryoelectron tomograms revealed that the nanotubes are in two different conformational states. Firstly, a primed state where the nanotube is assembled and embedded within the AS-7 cell envelope, beneath the S-layer. Second, an extended state where the nanotube elongates and traverses through the symbiont envelope and establishes cytoplasmic connection between the two cell types. The primed tubes were also present in host-only culture, but at much lower frequency. We interpret from this that the nanotube originates in the host and its biogenesis is upregulated in the presence of the symbiont. A composite average of the extended tubes supports our hypothesis that the extended nanotubes penetrate into the ARM-1 cytoplasm, supporting the notion that these tubes represent a mechanism for exchange of cytoplasmic components between the host and symbiont. However, limited resolution in this region should caution the readers interpretation. Although primed nanotubes are structurally aligned to the S-layer pores, the diameter of the tubes is larger compared to the S-layer pore size, suggesting large-scale structural rearrangement and/or symmetry breakage in the S-layer pore is essential for the tube extension. Intriguingly, we discovered a large barrel-shaped structure in the ARM-1 cell envelope that acts as a docking platform for the incoming nanotube. The barrel-shaped structure is reminiscent of bacterial secretin-like assemblies but much larger in diameter (21 nm). The structure could be formed from AS-7 proteins secreted through the tubes that assemble in the ARM-1 envelope. Considering the symbiotic nature of the interaction, an alternative hypothesis is that ARM-1 proteins could coordinate with the extended tube to build this docking platform. The protein components that comprise the main tube structure, and any accessory proteins are the subjects of ongoing research.

Employing Cryo-ET and STA, we were able to determine a 1 nm resolution in situ structure of the host S-layer, where domains of S-layers proteins were distinctly visible. The in situ structure of the AS-7 S-layer revealed a similar architecture to previously characterized S-layers from Sulfolobus islandicus, a related Archaea of the same family⁴³. The AS-7 host S-layer constitutes an array of dome-like structures with a pore in the center. Nanotube extension could happen through this pore but would require conformational rearrangement as the tubes are larger in diameter. Different conformations of SlaA have been reported in Sulfolobus spp. that alter the S-layer lattice in response to the environment⁴³. However, conformational changes that enlarge the pore are yet to be demonstrated in situ.

DPANNs are characterized by their small size and small genomes (0.5–1.5 Mb), which often lack genes for essential metabolic processes such as amino acid and nucleotide synthesis^2,44,45,46. The genome of ARM-1 was previously shown to be deficient in genes for glycolysis, gluconeogenesis, carbon and nitrogen fixation, and the biosynthesis of amino acids and nucleotides¹⁴. The reliance of ARM-1 and other DPANNs on their hosts is therefore evident, whereas the benefit to the host cell in this symbiotic relationship is less obvious. The ARM-1 AS-7 co-culture system provides an example of a host committing substantial resources and adaptations to facilitate interaction with a DPANN, which places new emphasis on the importance of this interaction for the host cell.

STA of the nanotube showed an inner lumen diameter of 4.4 nm, which is sufficiently wide to allow for folded larger globular proteins (100–150 kDa), DNA, and metabolites to pass^47,48. DPANN has been suggested to exchange nutrients, including amino acids, nucleotides, and sugars (for cell wall synthesis and metabolism). Confirmation of the molecular identity of the nanotube apparatus and substrates passing through the tube is difficult in our genetically intractable systems. Therefore, we utilized proteomics approaches to investigate molecular players involved in the AS-7 and ARM-1 symbiosis. Our proteomics analysis indicated that CO₂, TCA cycle, and amino acid metabolic pathways were upregulated in the host, these data suggest that metabolites from these processes are the intended cargos. In addition, CRISPR Cas9 proteins were upregulated in the host and DPANN during co-culture, a locus containing three hypothetical proteins was shown to be an operon associated with a CRISPR Cas9 system (BCS94299…BCS942301) (Supplementary Data 1). A recent study has uncovered the important role that CRISPR systems have in host-DPANN symbiosis; it is, therefore, plausible that the tubes may facilitate the transfer of DNA⁴⁹.

Cryo-ET can provide high-resolution cell biology insight into an otherwise inexplorable group of organisms. We surveyed the ARM-1 cells for cellular structures that may prove important for understanding their biology and the nature of their symbiosis, our results are presented as a gallery of features that we believe will be of importance to other researchers in the DPANN field (Supplementary Fig. 10). In addition, membrane tubes (as opposed to the protein tubes discussed above) connecting DPANN cells were observed in some tomograms that are suggestive of intercellular nanotubes. Intercellular nanotubes/nanopods have been shown in archaeal cells before; however, their function remains enigmatic. DPANN archaea are co-located on the host cell, creating a micro-community. Nanotube formation is a possible method to exchange nutrients within the DPANN community.

The world of DPANNs remain largely unexplored, their small genomes and obligate symbiotic lifestyles make their biology a fascinating area of research. The nature and evolution of these relationships are complex, and as we have shown here, the host can spend substantial resources to establish interaction and exchange biological molecules with the DPANN. At least in our system host-DPANN relationship appears to be mutualistic and ectosymbiotic. Our work demonstrates the capability of cryo-ET and STA to interrogate these genetically intractable systems and contributes to the expanding knowledge of DPANN biology and their symbiotic relationships.

Methods

Cultivation conditions and growth monitoring

A co-culture system composed of Microcaldus variisymbioticus strain ARM-1 and Metallosphaera javensis AS-7 was established as described previously¹⁴. The co-culture system, as well as the pure culture of M. javensis were cultivated at 60 °C under aerobic conditions. The growth of both strains was monitored by qPCR following the method as described previously¹⁴.

Cultivation conditions for cryoTEM

Cultures of Metallosphaera javensis AS-7 and a co-culture system composed of Microcaldus variisymbioticus ARM-1 and its host Metallosphaera javensis AS-7 were routinely cultivated at 55 °C using modified Brock’s basal salt medium⁵⁰ supplemented with 1 g/L yeast extract (MBSY medium) as described previously¹⁴. The pH of the medium was adjusted to 3.0 by the addition of 50% (v/v) sulfate. When the growth entered the stationary phase, the co-culture was filtered by a sterilized syringe filter (pore size: 0.45 μm, polyethersulfone). A 2 mL aliquot of the filtrate containing approximately 10⁷ cells of ARM-1 was added to 8 mL of a MBSY medium, followed by inoculation with 50 μL of pure AS-7 culture. This co-culture system was incubated at 60 °C under aerobic conditions in triplicate. After 5 days of incubation, the co-culture was used for cryo-EM, 200 μL aliquots of the cultures were regularly sampled for growth monitoring by quantitative PCR analysis.

Growth monitoring by qPCR

To monitor the growth of ARM-1 and AS-7, qPCR was conducted following the same method as described previously ¹⁴. Briefly, microbial DNAs of the cultures were extracted by a DNA extraction machine (magLEAD 6gC; Precision System Science) with DNA extraction reagent (MagDEA Dx SV; Precision System Science), qPCR was done with specific primers targeting the 16 S rRNA gene of each species.

Cultivation conditions for proteomics and scanning electron microscopy

A co-culture prepared by the same procedures described above and pure AS-7 culture were pre-incubated at 55 °C for 10 days. A 1 mL aliquot of the co-culture or pure AS-7 culture was inoculated into 100 mL of MBSY medium and incubated at 60 °C under aerobic conditions. A total of 6 sets of 100 mL scale cultures were prepared. After five days of incubation, these cultures (100 mL × 6) were used for proteomics, whereas 5 mL from one of the co-cultures was sampled and used for scanning electron microscopy.

Scanning electron microscopy

Microbial cells of the co-culture were fixed by 1% glutaraldehyde for 15 min at room temperature. A 5 mL of the fixed co-culture was filtered on a polycarbonate membrane filter with 0.1 μm pores (VCTP, 25 mm, Merck Millipore). Cells trapped on the filter were washed by passing through 500 μL of pure water five times. After washing, the filter was flash-frozen, dehydrated, and sputter-coated with osmium by the method described previously¹⁴. The sputter-coated sample was analyzed at an accelerating voltage of 4 kV under the scanning electron microscope (JSM-7500F; JEOL).

Sample preparation for Proteomic analysis

Cell suspensions were adjusted to 4% SDS, 100 mM Tris pH 8.5 and boiled for 10 min at 95 °C. The protein concentrations were then assessed by bicinchoninic acid protein assays (Thermo Fisher Scientific), and 200 μg of each biological replicate was prepared for digestion using Micro S-traps (Protifi, USA) according to the manufacturer’s instructions. Briefly, samples were reduced with 10 mM Dithiothreitol for 10 mins at 95 °C and then alkylated with 40 mM iodoacetamide in the dark for 1 h. Samples were acidified to 1.2% phosphoric acid and diluted with seven volumes of S-trap wash buffer (90% methanol, 100 mM Tetraethylammonium bromide pH 7.1) before being loaded onto S-traps and washed 3 times with S-trap wash buffer. Samples were then digested with 2 μg of Trypsin (1:100 protease:protein) in Tetraethylammonium bromide pH 8.5 overnight at 37 °C before being collected by centrifugation with washes of 100 mM Tetraethylammonium bromide, followed by 0.2% formic acid followed by 0.2% formic acid/50% acetonitrile. Samples were dried down and further cleaned up using C18 Stage 1,2 tips to ensure the removal of any particulate matter^51,52.

Reverse phase liquid chromatography-mass spectrometry

C18 enriched proteome samples were re-suspended in Buffer A* (2% acetonitrile, 0.01% trifluoroacetic acid) and separated using a two-column chromatography setup composed of a PepMap100 C18 20-mm by 75-µm trap (Thermo Fisher Scientific) and a PepMap C18 500-mm by 75-µm analytical column (Thermo Fisher Scientific) using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific). Samples were concentrated onto the trap column at 5 µl/min for 6 min with Buffer A (0.1% formic acid, 2% DMSO) and then infused into an Orbitrap 480™ (Thermo Fisher Scientific) at 300 nl/minute via the analytical column. Peptides were separated by altering the buffer composition from 3% Buffer B (0.1% formic acid, 77.9% acetonitrile, 2% DMSO) to 23% B over 59 min, then from 23% B to 40% B over 10 min and then from 40% B to 80% B over 5 min. The composition was held at 80% B for 5 min before being returned to 3% B for 10 min. The Orbitrap 480™ Mass Spectrometer was operated in a data-dependent mode automatically switching between the acquisition of a single Orbitrap MS scan (300–1600 m/z, maximal injection time of 25 ms, an Automated Gain Control (AGC) set to a maximum of 300% and a resolution of 120 k) and 3 s of Orbitrap MS/MS HCD scans of precursors (normalize collision energy of 30%, a maximal injection time of 22 ms, a AGC of 75% and a resolution of 15k).

Proteomic data analysis

Identification and LFQ analysis was accomplished using MaxQuant (v2.0.2.0)⁵³ using the M. javensis AS-7 (NCBI GCA_021654415.1) and the Microcaldus variisymbioticus ARM-1 (NCBI GCA_021654395.1) proteomes with Carbamidomethyl (C) allowed as a fixed modification and Acetyl (Protein N-term), as well as Oxidation (M), allowed as variable modifications with the LFQ and “Match Between Run” options enabled. The resulting data files were processed using Perseus (version 1.6.0.7)⁵⁴ with missing values imputed based on the total observed protein intensities with a range of 0.3 σ and a downshift of 1.8 σ and statistical analysis undertaken using a two-tailed unpaired T test. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁵⁵ partner repository with the dataset identifier PXD045678.

Gene conservation analysis

Conservation analysis of up- and downregulated proteins was performed by tBLASTn of protein sequences against a curated set of Archaeal genomes from family Sulfolobaceae from the genome taxonomy database (gtdb.ecogenomic.org). These genomes were classified and checked for quality in a previous study⁵⁶. and a full list is provided in supplementary Data 2. Putative orthologues retrieved by tBLASTn were retained if they had ≥50% amino-acid identify and ≥75% coverage relative to the query, and the presence/absence matrix generated in R. Extended annotation of the host and symbiont protein-coding sequences (from genbank accessions AP024486.1 and AP024487.1) to assign COG categories and predicted pathways were completed with EGGNog mapper version 2.1.9 with no taxonomic restriction on orthologs, and the following thresholds: e value 0.001, score 60%, ID 40, query and subject coverage 40%.

Cryo-ET data collection

Co-cultures were mixed with 10 nm colloidal gold beads (Sigma-Aldrich, Australia), which had been precoated with 1% BSA. The co-culture-colloidal gold bead mixture was added to glow-discharged copper R2/2 Quantifoil holey carbon grids (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were blotted (under 100% humidity conditions) and plunge-frozen in liquid ethane (Vitrobot Mark IV, FEI Thermo Fisher Scientific). Grids were imaged using an FEI Titan Krios G4, 300 keV FEG transmission electron microscope (Thermo Fisher Scientific), micrographs were collected using a Gatan K3 Summit direct electron detector equipped with an energy filter set to 20 eV. Tilt series of cells were collected automatically between −60° to +60° at 2° intervals using the FEI Tomography 5 data collection software. The cumulative total dosage used was 100 e⁻ Å⁻², with a defocus of −6 μm and a pixel size of 3.39 Å.

Data processing, segmentation, and subtomogram averaging

Tilt series were processed with either IMOD-tomo3D-PEET (data used in segmentation, and extended tube STA) pipeline or EMAN2 (contracted tube STA, S-layer STA for host and DPANN) workflow. IMOD processing used 2K binned micrographs, which were reconstructed by Tomo3D into 600 nm thick tomograms in 2K. The reconstructions were further binned to 1K for particle picking and segmentation. Segmentations were performed with the help of https://ariadne.ai/. Briefly, deep convolutional neural networks (CNNs) were trained using the manually generated ground truth and refined until a good quality of segmentation was reached. Subsequently, automated segmentations in 3D were performed. The segmentations were proofread and polished by expert inspection using the open-source software Knossos (https://knossos.app/). For each segmentation class, meshes and binary Tiff masks were generated for subsequent rendering and visualization in Knossos and Blender (https://www.blender.org/). Picked particles were then extracted from the 2K reconstructed tomograms and averaged using PEET.

In EMAN2, an initial model of the AS-7 S-layer was generated by averaging 200 high-quality S-layer particles manually selected from five tomograms. The selected particles were extracted with a box size of 52 from the tilt series at x4 binning (13.56 Å/pix). The extracted particles were then used to create a de novo initial model of the AS-7 S-layer using the initial model generator program in EMAN2.

The de novo initial model of the AS-7 S-layer was used as a reference for template matching which yielded 10,711 particles from 72 tomograms. The particles selected by template matching were extracted from each tilt series with a box size of 52 at x4 binning (13.56 Å/pix). The sub-tomograms were contrast transfer function (CTF) corrected at the per-particle-per-tilt level during extraction. The sub-tomograms were then aligned and averaged, to obtain initial alignments, which were then used to re-extract particles from each tilt series with a box size of 208 without binning (3.39 Å/pix).

For particles corresponding to the primed state of the tube, located beneath the AS-7 S-layer, 797 particles were manually selected from 28 tomograms. The selected particles were extracted with a box size of 168 (3.39 Å/pix) and CTF corrected. A de novo model of the tube was obtained using 50 high-quality particles using the EMAN2 initial model generator.

For both S-layer and tubes (primed state) averaging, seven iterations of subtomogram refinement were used, consisting of three rounds of 3D particle orientation, two rounds of 2D subtilt translation, one round of subtilt translation & rotation, and one round of subtilt defocus refinement with either c1 or c6 symmetry applied. The threshold was set to exclude the worst 20% of particles at each iteration step.

Modeling fitting and refinement

The full-length sequence of S-layer protein A (SlaA) and B (SlaB) of Metallosphaera javensis AS-7 was predicted using AlphaFold v2.0⁵⁷. AlphaFold multimer was used to predict the homotrimer of SlaB. The predicted models were fitted and refined into the density map representing both SlaA and SlaB proteins. Flexible N-terminal residues of SlaA (1–27) and SlaB (1–40) were removed during density fitting. Following visual inspection, the predicted full-length model of SlaA was split into 4 units (U1: 28–373; U2: 374–506, 507–639; U3: 640–784, 785–919; U4: 920–1076, 1077–1230, 1231–1345) each containing one or more domains. A semi-automated approach was employed to fit and refine individual units in a stepwise manner into the ~10 Å resolution map of S-layer complex. Initially, the individual units were fitted and refined as rigid bodies using ‘matchpt’ and ‘collage’ tool, respectively from the Situs package^58,59. Fits were finally refined using RIBFIND and Flex-EM conjugate gradient minimization^60,61. For SlaB, using visual inspection, the bounding box corresponding to a single SlaB trimer was identified and extracted from the full map of the S-layer. Situs ‘matchpt’ program was used to fit SlaB trimer. For SlaA, adjacent hexamers around the threefold symmetry axis were fitted using Segger tool in ChimeraX⁶². Finally, symmetry copies of SlaA and SlaB were generated using ChimeraX⁶³. A combination of manual and automated rigid body fitting was employed only for domains in U4 due to low resolution.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support this study are available from the corresponding authors upon request. Subtomogram averages of the AS-7 S-layer and primed nanotube have been deposited in the EM Data Resource and are available under accession codes EMD-42578 (In situ structure of AS-7 S-layer) and EMD-42579 (In situ structure of AS-7 primed tubes). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD045678 (Proteomics analysis of host AS-7 cells interacting with DPANN ARM-1).

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Acknowledgements

This project was funded by an NHMRC grant (APP1196924 to DG), an HFSP grant (RGEC33/2023) to DG and HDS, The Japan Society for the Promotion of Science through Grants-in-Aid for Scientific Research (21K15153 and 24K18201 to HDS). DCS is supported by the Melbourne Research Scholarship. We are thankful to Zhenan Li for their help with data processing. N.E.S. is supported by an Australian Research Council Future Fellowship (FT200100270) and an ARC Discovery Project Grant (DP210100362). Cryo-EM data were collected at the Ian Holmes Imaging Centre, Bio21, University of Melbourne. We thank the Melbourne Mass Spectrometry and Proteomics Facility of The Bio21 Molecular Science and Biotechnology Institute for access to MS instrumentation.

Author information

Hiroyuki D. Sakai
Present address: Japan Collection of Microorganisms, RIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan
These authors contributed equally: Matthew D. Johnson, Doulin C. Shepherd.

Authors and Affiliations

Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia
Matthew D. Johnson, Doulin C. Shepherd, Manasi Mudaliyar & Debnath Ghosal
ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia
Matthew D. Johnson, Doulin C. Shepherd, Manasi Mudaliyar & Debnath Ghosal
Department of Science and Engineering for Sustainable Innovation, Faculty of Science and Engineering, Soka University, Hachioji, Tokyo, 192-8577, Japan
Hiroyuki D. Sakai & Norio Kurosawa
Heart and Lung Research Institute, Department of Medicine, University of Cambridge, Cambridge, UK
Arun Prasad Pandurangan
Department of Microbiology and Infection Program, Biomedicine Discovery Institute, Monash University, 19 Innovation Walk, Clayton, VIC, 3800, Australia
Francesca L. Short
Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Parkville, VIC, Australia
Paul D. Veith
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
Nichollas E. Scott

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Matthew D. Johnson
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Contributions

M.D.J., H.D.S., and D.G. conceived the study. M.D.J. collected tomography data. M.D.J., D.C.S., M.M., and D.G. processed and analyzed cryo-ET data. D.C.S., M.D.J., and D.G. performed subtomogram averaging. H.D.S. and N.K. prepared co-culture samples. A.P.P. performed pseudo-atomic model fitting and analysis. N.E.S., M.D.J., and P.D.V. did proteomic data collection and analysis. F.L.S., M.D.J., and D.G. did comparative genomics analysis. M.D.J., H.D.S., and D.G. wrote the manuscript with input from all authors.

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Correspondence to Debnath Ghosal.

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Johnson, M.D., Shepherd, D.C., Sakai, H.D. et al. Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system. Nat Commun 15, 7066 (2024). https://doi.org/10.1038/s41467-024-51159-2

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Received: 29 March 2024
Accepted: 26 July 2024
Published: 16 August 2024
DOI: https://doi.org/10.1038/s41467-024-51159-2

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