Gene Duplication of the Bacterium Parasitizing the Nucleus of Mollusk Cells

A series of bacteria, cell have adapted to intracellular parasitism, but living within the cell nucleus is exceedingly rare for “high-level parasitism.” Nuclear parasitism is typically carried out by protists via pathogenic bacteria; however, a new study published in Nature Microbiology has identified such a phenomenon in deep-sea snails. ‘Ca. Endonucleobacter’ bacteria live in the gills of the bivalves Bathymodiolus puteoserpentis and Gigantidas childressi, divide within the cell nucleus, and reach populations exceeding 80,000. They induce “swelling” of organelles—the nucleus increases in size by 50-fold. Biologists have determined that the foundation of these unique adaptations lies in apoptotic inhibitor (IAP) factors acquired by the mollusk host genome, likely via viruses. Although horizontal gene transfer from eukaryotes to bacteria is considered rare, this new discovery adds to an increasing list of such examples.

Parasitism is a phenomenon notable for its universality. It has evolved across the most diverse levels of organization, from entire populations to individual cells, enabling organisms to exploit other life forms unilaterally. All “fully alive” viruses are intracellular parasites, mostly residing within cells including the nucleus. Previously, the lower limit of bacterial parasitism was thought to be within eukaryotic cells (such as mycoplasmas and mycobacteria) or other bacterial cells (like Bdellovibrio bacteriovorus). However, the discovery of bacteria living inside individual organelles—particularly nuclear parasites of protists—represents a novel “leap” in parasitism at previously unexplored scales. Nevertheless, bacteria parasitizing within mitochondria or other organelles are also known (J. Verbeke et al., 2023. Brucella abortus extracellular exit requires BNIP3L-mediated mitophagy).

Figure 1

Many marine organisms harbor gamma-proteobacteria from the family Endozoicomonadaceae. They are typically found in interstitial environments and adopt parasitic or commensal lifestyles; however, some members of this family live inside host cells. The first Endozoicomonadaceae were isolated from shrimp in 2007, followed by isolates from sponges, corals, and fish. To date, all cultivated members of this family in the laboratory are aerobic or facultative anaerobic cells with heterotrophic metabolic properties.

Nuclear parasitism by prokaryotes was previously known—primarily infecting protists but not animals (F. Husnik et al., 2021. Bacterial and archaeal symbioses with protists). A clade within Endozoicomonadaceae, ‘Ca. Endonucleobacter’ (Ca. or Candidatus indicating these bacteria are well-characterized but have not yet been cultured in the laboratory), infects the cell nuclei of deep-sea bivalve mollusks (Figure 1).

In a new study by researchers from the United States and Germany, tissues from Bathymodiolus puteoserpentis collected from hydrothermal vents on the Mid-Atlantic Ridge and Gigantidas childressi living in persistently low-temperature environments in the Gulf of Mexico were examined. Typically, the gill cells of these bivalve mollusks are infected with autotrophic bacteria that provide chemical nutrients to the host animals. The authors of the study identified a novel intranuclear parasitic bacterium from each mollusk species and assigned them the species epithets ‘Ca. Endonucleobacter puteoserpentis’ and ‘Ca. Endonucleobacter childressi’. Their sequences were assembled, complete genomes were obtained, transcriptomes and proteomes were characterized, and functional annotations were generated. Laser-capture microdissection combined with high-sensitivity RNA sequencing revealed interactions at different stages of infection. The localization of ‘Ca. Endonucleobacter’ was visualized using fluorescent in situ hybridization (FISH).

The researchers noted that the nuclear parasite never infects mollusk cells already hosting symbiotic bacteria. This holds true for both studied bivalve species, which represent different genera and distant aquatic regions, and is independent of the type of symbiont (G. childressi harbors only methanotrophic bacteria, whereas B. puteoserpentis harbors both methanotrophic and sulfide-oxidizing bacteria). Moreover, this pattern was also observed in specimens of the same mollusk species collected 13 years apart. It appears that the impossibility of parasitic infection of epithelial cells containing symbionts stems from surface modifications: these cells lack cilia and microvilli that parasites typically use to enter cells.

Additionally, the tissue distribution of the parasites in the two mollusks differs markedly (Figure 1). The gills of G. childressi contain nuclear parasites only in the outermost ciliated regions, whereas in B. puteoserpentis, the bacteria are evenly distributed throughout the gill tissues. The genomes of Endonucleobacter are relatively small and have low GC content. Metagenomic analysis revealed that the DNA of ‘Ca. Endonucleobacter puteoserpentis’ and ‘Ca. Endonucleobacter childressi’ contain significant differences; their genomes share only an average of 84.3% similarity. Phylogenetic analysis of 42 additional genomes showed that Endonucleobacter forms a monophyletic lineage, with its closest relatives belonging regularly to the family Endozoicomonadaceae (Figure 2).

Figure 2: ‘Ca. Endonucleobacter’ infection cycle.

A single ‘Ca. Endonucleobacter’ infects the host cell nucleus (early stage of infection), elongates and divides (mid-stage), and ultimately undergoes active division through septation of elongated cells (late stage). In the final stage, the mussel cell nucleus expands 50-fold and ruptures, releasing the parasites into the environment. a – ‘Ca. Endonucleobacter’ during early (E), mid (M), and late (L) infection stages. Middle panel: images obtained via FISH; lower panel: images obtained via transmission electron microscopy. Specific FISH stains show the localization of the parasite within the nucleus (DNA stained blue with DAPI) and symbiont-containing cells (indicated by dashed lines). Labels: e – ‘Ca. Endonucleobacter’; c – chromatin; ne – nuclear envelope. b – Phylogenetic analysis using 172 conserved marker genes derived from the genomes of ‘Ca. Endonucleobacter’ and its 42 relatives. The tree was constructed based on thousands of repeats. Image from the studied article.

The next important question is what these bacteria feed on, given their highly unusual ecological niche? They show no apparent nutritional deficiency—they grow to populations of up to 80,000 cells and stretch the nucleus 50-fold. Previously, it was hypothesized that bacteria living within the nucleus feed on nucleic acids (DNA and RNA) and chromatin. Based on the data obtained, the authors noted that Endonucleobacter exhibits a significantly higher growth rate than previously recorded and displays a characteristic enrichment profile. This growth is likely linked to the availability of dissolved nutrients and molecules. Moreover, previously known parasites have never been observed parasitizing in locations where host cells lack sufficient nutrients, such as the endoplasmic reticulum.

The newly discovered nuclear parasites not only reveal microbial diversity in marine environments for the first time but also provide significant new insights into how parasitism of eukaryotes by Endonucleobacter bacteria has evolved on a broader scale.