The parasitic lifestyle of an archaeal symbiont

Abstract

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.

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.

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.

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.

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 BMGE (alignment length, 11399 aa). Tree was inferred in IQ-TREE 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.