Dynamics of Ca. Nha. antarcticus—Hrr. lacusprofundi interactions
Our enrichment culture of the symbiont Ca. Nha. antarcticus and several Hrr. lacusprofundi host strains1 offers an ideal system for studying archaeal symbiosis. Importantly, these mixed cultures generate large numbers of nanohaloarchaeal cells (so that they make up ~50% of total cells in a co-culture at ~108 cells mL−1), which can be isolated, and used to infect a pure culture of a single host strain. In addition to the two species of interest, the enrichment culture also contains a low abundance (<1%) Natrinema species, which occupies a similar ecological niche to Hrr. lacusprofundi and has proven resistant to attempts to remove it from the culture1. To investigate Ca. Nha antarcticus—Hrr. lacusprofundi interaction dynamics, we used MitoTracker fluorescent dyes23 as vital cell stains to identify and track live interactions between Ca. Nha. antarcticus and Hrr. lacusprofundi strain R1S124 as well as electron microscopy to investigate morphological features.
For this analysis, purified Ca. Nha. antarcticus cells were stained with MitoTracker DeepRed (MTDeepRed) followed by incubation with MitoTracker Orange (MTOrange)-stained Hrr. lacusprofundi. Live co-cultures of labelled cells were then immobilised and cultured on an agarose gel pad or in a microfluidic flow chamber and imaged using time-lapse fluorescence microscopy, 3D laser scanning confocal microscopy, and 4D (3D time-lapse) live cell imaging. In agreement with previous work on other haloarchaeal species23, these Mitotracker dyes are retained by Hrr. lacusprofundi cells, and do not affect cell growth rates (Supplementary Figs. 1, 2 and 3), suggesting they are non-toxic.
A total of 163 MTOrange-stained Hrr. lacusprofundi cells were analysed in detail during two incubations over a period of 24 h on agarose pads. Of these, 132 cells (81%) were observed with one or more MTDeepRed-stained Ca. Nha. antarcticus cell(s) attached at the first timepoint imaged (0 h), indicating that attachment predominantly occurred during the initial incubation period (≤ 1 h) prior to commencement of time-lapse imaging (Supplementary Dataset 1). Over time, the fluorescent signal from Ca. Nha. antarcticus cells appeared to shift, so that over time more Ca. Nha. antarcticus cells appeared to be located within the bounds of their Hrr. lacusprofundi host (Fig. 1, Supplementary Fig. 4). Confocal imaging with 3D-orthogonal projection after 10 h incubation showed discrete regions within Hrr. lacusprofundi cells positive for MTDeepRed, suggestive of infiltration of the host cytoplasm by Ca. Nha. antarcticus (Fig. 1c, Supplementary Fig. 5). The migration of fluorescent signal from nanohaloarchaeal cells inside the boundary of the host appeared to take several hours, but the exact duration varied between different observed interactions (Fig. 1, Supplementary Fig. 4). Once present within the bounds of the Hrr. lacusprofundi cell, the area occupied by the Ca. Nha. antarcticus dye cell expanded over time (Fig. 1a, Supplementary Figs. 4 and 6).
Fig. 1: Live fluorescence and qPCR support Ca. Nha. antarcticus entering Hrr. lacusprofundi cells and causing lysis.
a A representative live fluorescence time-lapse series of Ca. Nha. antarcticus cells (MitoTracker DeepRed, coloured Magenta) attached to a host Hrr. lacusprofundi cell (MitoTracker Orange, coloured Green) (0–9 h), migrating internally (~10–21 h), followed by lysis of the host (22 h). b, qPCR quantification of 16S rRNA gene copy numbers from both organisms show active replication of Ca. Nha. antarcticus (Magenta circle) during the first 12 h of incubation followed by an ~73% decrease in Hrr. lacusprofundi (Green square: Co-culture Hrr. lacusprofundi, Blue triangle: Pure Hrr. lacusprofundi) 16S rRNA gene copy number between 12 h and 24 h. A second decrease in Hrr. lacusprofundi 16S rRNA gene copy number is seen between 48 h and 62 h resulting in a ~ 99% decrease in Hrr. lacusprofundi 16S rRNA gene copy number within co-cultures across the entire 62 h incubation compared to ~26% in the pure Hrr. lacusprofundi control. Data are presented as the mean value ± the standard deviation across the qPCR reactions (n = 3 technical replicates, Source Data are provided as a Source Data file). c A 3D confocal orthogonal slice images (left) and z-slices (right) of Ca. Nha. antarcticus cells appearing internalised within Hrr. lacusprofundi after 10 h incubation. Scale bars: a – 1 µm, c – 500 nm.
Over the course of the 24 h incubation period, 27% (36/132) of the Hrr. lacusprofundi cells that were observed with attached Ca. Nha. antarcticus cell(s) underwent lysis, accounting for 22% (36/163) of total Hrr. lacusprofundi cells in co-cultures (Fig. 1a, Supplementary Figs. 4, 5 and 6, Supplementary Dataset 1). Lysis occurred relatively rapidly and was complete within the 30 min time window separating image acquisitions. By contrast, no lysis was observed over periods of up to 70 h in the control samples of pure host cells (Supplementary Fig. 1, Supplementary Dataset 1). Upon lysis, the dye used to label host cells in co-cultures dissipated completely, whereas the label associated with Ca. Nha. antarcticus cells remained undimmed (e.g., Fig. 1a, compare 21 h and 22 h). These results are consistent with the survival of Ca. Nha. antarcticus cells following host cell lysis.
To investigate whether the timings of events observed in live fluorescence imaging corresponded to observable changes in the 16S rRNA gene copy number of each organism, samples were taken from a co-culture at regular timepoints, and DNA was extracted for qPCR. This revealed a ~10-fold increase in the estimated copy number of Ca. Nha. antarcticus 16S rRNA gene copy between 0 and 12 h, indicative of active replication in co-cultures (Fig. 1b). This was accompanied by a moderate decrease in Hrr. lacusprofundi 16S rRNA gene copy number (~30%, 0–12 h). Then, between 12 and 24 h, Hrr. lacusprofundi 16S rRNA gene copy number decreased by an additional ~73%, leading to a total decrease of ~81% in 16S rRNA gene copy number over the first 24 h. Following this, 16S gene copy numbers stabilised somewhat, before copy number from both organisms displayed a decrease between 48 h and 62 h (Ca. Nha. antarcticus: ~42% decrease, Hrr. lacusprofundi: ~97% decrease). In total, over the 62 h incubation, Ca. Nha. antarcticus 16S rRNA gene copy number increased 3.4-fold, while Hrr. lacusprofundi 16S rRNA copy number decreased ~99%. Over the same time period, 16S rRNA gene copy number decreased ~25% in control pure cultures of Hrr. lacusprofundi. These data suggest that the interaction of the symbiont with the host is parasitic rather than mutualistic.
To assess replicability and specificity of apparent internalisation, 16S rRNA targeted FISH microscopy with addition of a lectin cell surface stain (Concanavalin A conjugated with Alexa Fluor 350 (ConA-AF350)) was conducted on samples of co-cultures incubated for 16 h (Fig. 2a, b). A Z-stack of a Hrr. lacusprofundi cell co-fluorescent for the Ca. Nha. antarcticus 16S rRNA probe shows nanohaloarchaeal 16S rRNA signal localised within the host cell, and within the region bounded by ConA-AF350. These data support the idea that the cytoplasmic contents of these nanohaloarchaeal cells have entered the host (Fig. 2a, b). To further test whether the translocation of the Ca. Nha. antarcticus contents into the Hrr. lacusprofundi cells corresponded to the complete internalisation within a live host, ConA-AF350 was used together with a live-cell-impermeable stain (RedDot 2) to both label surface-bound nanohaloarchaeal cells and to assess loss of host cell membrane integrity, respectively. As expected, ConA-AF350 added to co-cultures labelled Ca. Nha. antarcticus cells that were attached to the surface of Hrr. lacusprofundi (Fig. 2c, Supplementary Fig. 7). By contrast, when ConA-AF350 was added to co-cultures at later time-points (6 h), many foci positive for the Ca. Nha. antarcticus dye did not appear positive for ConA-AF350 (Fig. 2d, Supplementary Fig. 8), consistent with their complete internalisation within host cells. At the same time, the absence of RedDot 2 staining indicated that host cells remained intact during the internalisation process (Fig. 2d, Supplementary Fig. 8). Conversely, host cells that were inferred to have lysed via the loss of MitoTracker Orange signal stained positive for RedDot 2 as expected (Fig. 2e, Supplementary Fig. 8). Over the course of 0–6 h incubations, the proportion of Hrr. lacusprofundi lysis events associated with Ca. Nha. antarcticus cells increased from ~23% to ~80%, while the proportion of nanohaloarchaeal cells attached to a host cell increased from ~6% to ~41% (Fig. 2f, g, Supplementary Dataset 2). Throughout, Ca. Nha. antarcticus cells associated with lysed Hrr. lacusprofundi cells labelled positive for both MitoTracker and Concanavalin A stains (Fig. 2e, Supplementary Fig. 9).
Fig. 2: Cell surface stains support the internalisation of Ca. Nha. antarcticus material.
a and b Fluorescence micrographs of a co-culture of Ca. Nha. antarcticus (Table 1. Nha_FISH_Probe, coloured Magenta), Hrr. lacusprofundi (Table 1, Hrr_FISH_Probe, coloured Green), and cell surface (ConA-AF350, coloured Blue). a Orthogonal projection of Hrr. lacusprofundi cell with signal for the Ca. Nha. antarcticus FISH probe inside the bounds of the host cell. b, Individual channels and composite image of z-slice from stack used to produce projection in (a). c–e Live fluorescence micrographs taken 6 h post-mixing showing Ca. Nha. antarcticus (MitoTracker Green, coloured Magenta) interactions with Hrr. lacusprofundi (MitoTracker Orange, coloured Green) including additional stains for cell surface (ConA-AF350, coloured Blue), and cell death (RedDot 2, coloured red). c Representative fluorescence micrographs showing Ca. Nha. antarcticus cells (MitoTracker Green, coloured Magenta) attached to the surface of Hrr. lacusprofundi (MitoTracker Orange, coloured Green). Cell surface staining (ConA-AF350, coloured Blue) shows foci corresponding to regions where Ca. Nha. antarcticus was attached to the host cell. No signs of lysis were detected by a dead cell stain (RedDot 2, coloured Red). d Representative live fluorescence micrographs showing Ca. Nha. antarcticus cells (stained with MitoTracker Green, represented Magenta) that appear internalised within Hrr. lacusprofundi cells (stained with MitoTracker Orange, represented Green). Cell surface staining (Concanavalin A, represented Blue) does not show foci corresponding to Ca. Nha. antarcticus cells, indicating the surface of the symbiont is inaccessible to the dye. No signs of lysis are evident from inclusion of a dead stain (RedDot 2, represented Red). e Representative fluorescence of Hrr. lacusprofundi (MitoTracker Orange, coloured Green) lysis events associated with Ca. Nha. antarcticus (MitoTracker Green, coloured Magenta). Lysis is indicated by positive fluorescence for RedDot 2 (coloured Red) and is associated with loss of MitoTracker Orange signal from the host cell while the Ca. Nha. antarcticus cells remain intact and positive for both MitoTracker Green and the cell surface stain (Con-AF350A, coloured Blue). Quantitative data for (f) lysis and (g) attachment events over short-term incubations. Data show (f) percentage of lysis events associated with a Ca. Nha. antarcticus cell and (g) the percentage of Ca. Nha. antarcticus cells attached to host cells over the course of a time series (0–6 h). Data show average number of events across triplicate experiments, and error bars represent standard deviation as summarised in Supplementary Dataset 2. Arrows: examples of Ca. Nha. antarcticus cells; Scale bars: a, b: 1 µm, c–e: 500 nm.
To complement this analysis, similar experiments were performed using continuous liquid flow culture (in a microfluidics system) to assess the interactions of immobilised, MTOrange stained Hrr. lacusprofundi R1S1 cells (0.7–1.1 μm trap height) with MTDeepRed stained, FACS-purified, Ca. Nha. antarcticus cells (Supplementary Fig. 6, Supplementary Movie 1). As with the agarose pad experiments, Ca. Nha. antarcticus cell(s) attached to Hrr. lacusprofundi cells before the first images could be observed. Again, over a 2–23 h time-period, the presence of the internalised Ca. Nha. antarcticus MTDeepRed signal was associated with decreased signal intensity and increased area within the host (Supplementary Fig. 6). It was notable that during the time course, 360 Hrr. lacusprofundi cells lysed in the infected culture (56%), whereas no lysis occurred in the uninfected and unstained control (407 cells), and only two lysis events occurred in the uninfected and stained control (654 cells) (Supplementary Fig. 2 and 6, Supplementary Dataset 1). Taken together, these agarose pad and microfluidic experiments demonstrate that Ca. Nha. antarcticus cells induce lysis of their hosts (22–56% of total Hrr. lacusprofundi cells in the infected cultures were lysed versus ~0% in the control (Supplementary Dataset 1)).
Experiments were also performed to investigate the effect of Ca. Nha. antarcticus on the morphology of Hrr. lacusprofundi, which includes rods, disks, and coccoid cells (Supplementary Fig. 2, Supplementary Dataset 1 and ref. 25,26). After co-incubation with MTDeepRed-stained Ca. Nha. antarcticus cells, 34% rod-shaped MTOrange-stained Hrr. lacusprofundi cells (on agarose pad) had become more rounded (Supplementary Figs. 4 and 10). This morphological change was not seen for control Hrr. lacusprofundi cells (Supplementary Figs. 1 and 10, Supplementary Dataset 1). A higher proportion of such morphological changes of co-cultured Hrr. lacusprofundi compared to pure culture was also seen with the microfluidics experiments (Supplementary Dataset 1). These results suggest that the association of the two species impacts the structure of the host cell envelope or the arrangement of S-layer proteins of the host cell.
Structural features of the Ca. Nha. antarcticus symbiosis
To investigate the structural features of cells in which nanohaloarchaeal cytoplasmic contents labelled with MTDeepRed were seen within the bounds of a host Hrr. lacusprofundi cell, we performed cryo-correlated light and electron microscopy (cryo-CLEM) followed by electron cryo-tomography (cryo-ET). Cells were fluorescently labelled as before and incubated together for 16 h to enable attachment and invasion prior to vitrification by plunge-freezing and imaging. This timepoint was chosen to maximise the chances of observing cells within their hosts. When imaging the fluorescent stain using CLEM, we looked for Hrr. lacusprofundi cells that were co-labelled for the MTDeepRed used to stain Ca. Nha. antarcticus. This identified examples in which MTDeepRed fluorescence was confined to discrete regions of the host cell or was present throughout the host cell (Fig. 3, Supplementary Fig. 11, Supplementary Movies 2 and 3). Cryo-ET of the subset of cells that possessed localised fluorescent signals from MTDeepRed revealed internal membranous structures at locations where MTDeepRed signal was present (Fig. 3). Similar structures could also be identified in cells with diffuse MTDeepRed signal (Supplementary Fig. 11).
Fig. 3: Cryo-correlative light and electron microscopy of an internal structure within a Hrr. lacusprofundi cell from a Ca. Nha. antarcticus – Hrr. lacusprofundi co-culture.
a Cryo-fluorescence microscopy images show a Hrr. lacusprofundi cell (stained with MitoTracker Green, coloured green) with signal consistent with internalisation of Ca. Nha. antarcticus cytoplasm (stained with MitoTracker DeepRed, coloured Magenta). b Cryo-TEM micrograph of the same cells shown in (a) used for identification of regions for tomography. c Overlay of cryo-fluorescence and cryo-TEM images. Z-slices from tomogram of internalised structure showing d full field of view and e internal structure. The cell envelope of the internal structure appears to possess multiple additional layers compared to non-internalised nanohaloarchaeal cells (Supplementary Fig. 14a–d). Due to logistics of equipment access these experiments were performed once. Scale bars: a 5 µm, b, c 500 nm, d, e 100 nm.
To investigate the ultrastructure of Hrr. lacusprofundi and Ca. Nha. antarcticus in greater detail, higher quality three-dimensional images of Hrr. lacusprofundi and Ca. Nha. antarcticus cells were acquired for both pure samples and co-cultures using cryo-ET. Ca. Nha. antarcticus cells observed in pure samples and in co-cultures that were external to Hrr. lacusprofundi cells possessed structures resembling a classical archaeal cell envelope structure with a single lipid bilayer and S-layer. Consistent with the cryo-CLEM data, internal membrane-bound structures (~80–250 nm diameter) were observed in several cryo-ET samples of Hrr. lacusprofundi cells incubated with Ca. Nha. antarcticus cells (Supplementary Figs. 12 and 13, Supplementary Movies 4−8). In some cases, internal structures were visible in intact host cells as visualised by scanning in z (Supplementary Figs. 12a–c, 13c, d)—consistent with the idea that the structures formed within Hrr. lacusprofundi cells can occur without inducing host cell lysis. At the same time, internal structures were also seen in cells that appeared damaged or with a disrupted outer membrane (Supplementary Figs. 12d–f, 13a, b). In both cases, the internalised structures were highly radiation sensitive, similar to the fluorescently labelled structures observed with cryo-CLEM, limiting the achievable resolution of the images. Nevertheless, in many instances, the internal membrane-bound structures seen in co-cultures had a surface that exhibited a repeating pattern16,27 characteristic of an S-layer (Supplementary Fig. 12b). Archaea and Bacteria are known to possess several mechanisms to prevent S-layer proteins assembling in the cytoplasm, indicating that these repeating structures are unlikely to constitute the host S-layer28,29,30. The presence of internal membranes and a putative S-layer within infected hosts suggests that these features may represent intact Ca. Nha. antarcticus cells or material derived from Ca. Nha. antarcticus cells (see Supplementary Discussion: Internal Membrane-bound Structures).
In pure Ca. Nha. antarcticus samples, cells exhibited bulges within the membrane and cytoplasmic structures (Supplementary Fig 14a–d, Supplementary Movies 9−12). In appearance, these cytoplasmic structures in Ca. Nha. antarcticus, which possess a surface monolayer surrounding a higher electron density core and uniform texture, resemble polyhydroxyalkanoate-like (PHA-like) granules previously identified in Hrr. lacusprofundi31. The bulges within the Ca. Nha. antarcticus membrane resemble lipid droplets32. It is notable that similar structures were also observed in the membranes of Hrr. lacusprofundi cells interacting with Ca. Nha. antarcticus cells (Supplementary Fig. 12d–f and 14e–h, Supplementary Movies 6, 13, 14) but were not observed in the membranes of uninfected Hrr. lacusprofundi cells (Supplementary Fig. 14i–k, Supplementary Movies 15−17), suggesting that the Nanohaloarchaeota play a role in inducing their formation. This is potentially significant as Ca. Nha. antarcticus lacks identifiable genes for both lipid biosynthesis and metabolism1 and must therefore acquire bulk lipids from the host to survive.
We also used Cryo-ET to examine the contact sites between Ca. Nha. antarcticus and Hrr. lacusprofundi cells prior to invasion (Supplementary Fig. 14e–h, Supplementary Movies 13 and 14). In some cases, these images suggested disruption of the two cell membranes and the opening of a cytoplasmic channel—similar to the structure of the interaction interface previously reported for N. equitans and I. hospitalis cells19. In these cases, a gap is visible in the S-layers of both organisms at the interaction site (Supplementary Fig. 14e–h, Supplementary Movies 13 and 14). In all cases, despite the close physical association of the two cells, this region of close membrane apposition was limited to a section of ~15–20 nm in width.
Candidate genes mediating interactions
In an effort to better understand the molecular mechanisms allowing Ca. Nha. antarcticus to engage in this peculiar case of archaeal parasitism, in this study we also took a closer look at the Ca. Nha. antarcticus genome. Type IV pili are believed to play an important role in the lifestyles of Bdellovibrio bacteriovorus33, Candidatus Vampirococcus lugosii34, Ca. Saccharibacteria TM7i35, and may also facilitate interactions between Ca. Nha. antarcticus and Hrr. lacusprofundi. Analysis of a set of 569 representative archaeal genomes revealed the presence of two conserved loci encoding Type-IV pilus homologues across multiple cluster 2 DPANN8 lineages (Nanohaloarchaeota, Woesearchaeota, Pacearchaeota, Nanoarchaeota, and Aenigmarchaeota) as well as Undinarchaeota (Fig. 4). In addition to Type-IV pilus genes, these loci also encode proteins with coiled-coil domains predicted to structurally resemble viral attachment proteins (sigma-1 protein: PDB_ID 6GAO, Fig. 4, Supplementary Figs. 15–23). Since similar loci are also present in a cultivated species of Nanoarchaeota (Ca. Nanoclepta minutus13), which has not been reported to induce similar internal structures as Ca. Nha. antarcticus, it remains to be investigated whether they are involved in forming the structures observed in the system studied here. Previously generated proteomics data1 confirmed several proteins within the Ca. Nha. antarcticus loci are actively synthesised, including the coiled-coil domain containing proteins. In addition to the Type-IV pilus-like loci, comparisons of the genetic content between Ca. Nha. antarcticus and Ca. Nanohalobium constans17 (a cultivated nanohaloarchaeon that was not reported to invade host cells) revealed that Ca. Nha. antarcticus encodes proteins that structurally resemble autolysins, bacteriocins, and phage cell-puncturing proteins (Supplementary Discussion), which are absent from the Ca. Nanohalobium constans genome, suggesting the possibility that they may support predation of the host.
Fig. 4: Conservation of loci encoding CCP genes in Nanohaloarchaeota.
a A maximum-likelihood phylogenetic tree based on 51 marker proteins and 569 archaeal species. The alignment was trimmed with BMGE61 (alignment length, 11399 aa). Tree was inferred in IQ-TREE81 with the LG + C20 + F + R model with an ultrafast bootstrap approximation (left half of bootstrap symbol) and SH-like approximate likelihood test (right half of bootstrap symbol), each run with 1000 replicates (see key for shading indicating bootstrap support). The tree was artificially rooted between DPANN Archaea (cluster 1 DPANN in dark purple, cluster 2 DPANN in green) and all other Archaea (shaded in grey). The number of species represented in each clade is shown in parentheses after the taxonomic name of the clade. Scale bar: average number of substitutions per site. b OmegaFold predicted coiled-coil structures of both the Ca. Nha. antarcticus locus 2 CCPs (NAR1_03220 and NAR1_01690). c The two Cluster 2 DPANN loci are shown aligned to the Nanohaloarchaeota sequences in the phylogenetic tree. Ca. Nha. antarcticus proteins identified in proteomic data are highlighted (bold outline). The type-IV filament proteins encoded in each locus (CpaF, pilus assembly ATPase; TadC, membrane assembly platform) or just Locus 1 (FlaF and PilA, filament proteins) are shown. Other proteins encoded in Locus 1 are Mpg (3-methyladenine DNA glycosylase), GroEL (chaperonin), GatE (Archaeal Glu-tRNA (Gln) amidotransferase subunit E) and NTPhyd (P-loop containing nucleoside triphosphate hydrolase). The gene-locus images were manually generated and loci were only included if they had putative flagella or pili genes up- or downstream of the CCP genes.
Ca. Nha. antarcticus is a parasitic archaeon
Our data demonstrates that the relationship between Ca. Nha. antarcticus and Hrr. lacusprofundi is parasitic, with interactions between the two organisms leading to lysis of a large proportion of host cells. This likely explains why co-cultures of the two organisms cannot be stably maintained1. Several lines of evidence also show that either entire nanohaloarchaeal cells or nanohaloarchaeal cytoplasmic contents enter the host, prior to host cell lysis. First, fluorescence microscopy approaches demonstrate that initial attachment of nanohaloarchaeal cells is followed by internalisation of the nanohaloarchaeal MTDeepRed signal, so that the signal from the nanohaloarchaeal cell eventually appears diffuse and fully encapsulated within the host cell (Fig. 1, Supplementary Fig. 4). Second, whereas externally attached nanohaloarchaeal cells can be labelled using a surface dye (ConA), the surfaces of nanohaloarchaeal cells that appear internalised based on the MTDeepRed signal are inaccessible to an external dye, (Fig. 2, Supplementary Fig. 8). Finally, using cryo-CLEM, we were able to visualise internal, membrane bound structures within infected Hrr. lacusprofundi cells at locations that were correlated with the presence of fluorescently labelled nanohaloarchaeal cells (Fig. 3, Supplementary Fig. 11). Taken together, these data suggest that Ca. Nha. antarcticus cells may invade the host cytoplasm during infection.
It should be noted that many of the internalised structures observed were smaller than free-living nanohaloarchaeal cells. Thus, it is possible that these internal structures arise from surface-bound nanohaloarchaeal cells, rather than from their complete internalisation. In the event these structures represent bona fide live internalised nanohaloarchaeal cells, the internalisation process may facilitate the acquisition of essential nutrients, including the lipids required for membrane formation and those forming lipid droplets in free Ca. Nha. antarcticus cells. Given the eventual loss of host cell integrity following infection, the internalised Ca. Nha. antarcticus cells could be released via host cell lysis. In some cases, however, Cryo-ET images of internal structures were suggestive of a multi-layered internalised envelope (Fig. 3), indicating that part of the host surface may be internalised as well as the symbiont cell—perhaps in a process akin to endocytosis, or via internal vesiculation like that observed in L-form bacteria36. While internalisation via an endocytosis-like event could enable a nanohaloarchaeal cell to enter a host without bursting it more easily than alternative entry mechanisms, it is possible that the apparent multi-layered envelopes observed represent artifacts of electron damage, which can affect tomographic reconstructions (Fig. 3). The dose sensitivity of the sample limited our capacity to determine which of these possible explanations is most likely. Thus, further work will be necessary to confirm the putative internalisation of Ca. Nha. antarcticus and determine how this may be achieved.
The observed activity of Ca. Nha. antarcticus shows similarities to the recently reported lifestyle for Ca. V. lugosii, a CPR bacterium recently reported to prey on a gammproteobacterium34. However, Ca. V. lugosii does not appear to invade its host cells but remains in an ectosymbiotic state34. The apparent internalisation of nanohaloarchaeal cytoplasmic contents (either via intact cells or vesicles) also bears some similarities to viruses and some bacterial predators, most notably B. bacteriovorus33. While there are examples of archaeal endosymbionts of eukaryotes (e.g. methanogenic protist endosymbionts37,38), Archaea have not previously been shown to enter other archaeal cells, to host intracellular symbionts, or to induce internal vesiculation in symbiotic partners.
In describing a parasitic DPANN archaeon whose interactions with its host results in host cell lysis, this work adds to a growing number of examples of species across both Bacteria and Archaea with the capacity to impact community structure through host species predation34,39. It is unclear how widespread such parasitic lifestyles are amongst DPANN Archaea, as the majority of DPANN are uncultivated8 and the factors that influence growth of DPANN remain enigmatic. However, the observations we describe in this paper illustrate the potential capacity of certain DPANN Archaea to contribute to nutrient cycling through lysis of host cells, similar to viral predation in the top-down control of the food web in Antarctic aquatic systems40. The lysis of host cells and release of organic material into the environment by Ca. Nha. antarcticus is likely to increase supply of organic and inorganic nutrients to the wider microbial community. This may, in turn, stimulate growth of diverse members of the community and prevent sequestration of nutrients within host cells. In this way, Ca. Nha. antarcticus is likely to contribute to the recycling of nutrients in the three haloarchaeal-dominated, hypersaline lakes that it is known to colonise1. Considering that it has been suggested that DPANN Archaea may associate not only with other archaea but also with Bacteria7,9,41,42, the capacity of some representatives of the DPANN Archaea to behave in such a predatory manner could have implications for microbial food web dynamics across the globe and may necessitate a re-evaluation of their functional importance and ecological roles.
