Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Abstract

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named 'Candidatus Nucleicultrix amoebiphila', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. 'Candidatus Nucleicultrix amoebiphila' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96-120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.

Figure 1

The intracellular niche of ‘Candidatus Nucleicultrix amoebiphila'. Transmission electron micrographs showing different infection stages of Hartmannella sp. (a–d) and A. castellanii (e–h). (a) Early infection stage in Hartmannella with three Nucleicultrix particles present in the nucleus, bacteria are surrounded by host electron-dense heterochromatin. (b) Intermediate infection stage; the nucleus is filled with bacteria. (c) Late infection stage with an enlarged nucleus. (d) Intact cyst containing symbionts; the nuclear membrane is ruptured. (e) Early infection stage in A. castellanii; after uptake into the cytoplasm, the bacteria remain enclosed by a vacuole and are surrounded by an electron-translucent space indicating the presence of extracellular polymeric substances. (f) Symbionts in close proximity to the host nuclear membrane; no clear phagosomal membrane can be recognized. (g) Ultrastructure of the symbionts inside an Acanthamoeba sp. nucleus; coccoid rods with a Gram-negative-type cell wall can be seen, one dividing cell is shown. (h) Lysis of an A. castellanii host cell; the nucleus is densely populated by bacteria, while the nuclear membrane is still intact. Asterisks indicate ‘Candidatus Nucleicultrix amoebiphila' arrows indicate the nuclear membrane; m, mitochondria; n, nucleolus; hc, heterochromatin; v, vacuole. Bars, 1 μm.

Figure 2

Phylogenetic relationship of ‘Candidatus Nucleicultrix amoebiphila' with the Alphaproteobacteria. (a) Maximum likelihood tree based on a concatenated 16S–23S rRNA alignment. ‘Candidatus Nucleicultrix amoebiphila' (indicated by an asterisk) together with other protist symbionts forms a deeply branching lineage within the Alphaproteobacteria. (b) 16S rRNA-based maximum likelihood tree showing members of the protist symbiont clade containing ‘Candidatus Nucleicultrix amoebiphila'. Circles displayed at the nodes represent bootstrap values (upper half) and Bayesian posterior probability values (lower half), respectively. Black indicates support values >80% or probability values >0.8; gray indicates support values of 50–80% or probability values of 0.5–0.8, white indicates no support. Fully resolved trees are shown in Supplementary Figures S3–S5; accession numbers of sequences used for phylogenetic analyses are listed in Supplementary Table S1.

Figure 3

Infection cycle of ‘Candidatus Nucleicultrix amoebiphila' in Hartmannella sp. FS5. Infection was monitored over a course of 144 h and visualized with FISH using probes CBR125 (orange) and EUK516-Cy5 (gray). Single bacterium starts colonizing the nuclear compartment within the first 6 h post infection; the nucleus is completely filled with bacteria after 96 h. Fully infected and highly enlarged nuclei can be observed after 120 h. The course of infection of Acanthamoeba sp. cells is similar (Supplementary Figure S7). Bar, 5 μm.

Figure 4

Modes of transmission of ‘Candidatus Nucleicultrix amoebiphila'. Key events during transmission of Nucleicultrix in Hartmannella sp. visualized by FISH are shown in the left panel. Bacteria and amoebae were labeled with probes CBR125 (orange) and EUK516 (gray), respectively. A dividing Hartmannella trophozoite is shown (i) in which the distribution of Nucleicultrix among both daughter cells can be seen. Note the single bacterial cell (arrows) within the amoeba nucleus undergoing segregation. Host cell lysis (ii) occurs only rarely in Hartmannella sp.; bar, 10 μm. A model of the life cycle and transmission of Nucleicultrix in amoebae is shown in the right panel. Although vertical transmission is predominant in Hartmannella sp., host lysis and horizontal transmission is the major route of Nucleicultrix in acanthamoebae.

Figure 5

Fate of amoeba hosts during infection with ‘Candidatus Nucleicultrix amoebiphila'. (a, b) Viability of amoeba cells during infection with Nucleicultrix. PI fluorescence intensities indicating dead amoeba cells were determined during the course of infection. A. castellanii but hardly Hartmannella sp. cells are lysed in the presence of Nucleicultrix. The percentage of infected amoebae during the course of infection is shown by filled circles. (c, d) Amoeba cell numbers during the course of infection. Asterisks indicate significant differences to the control (P<0.05), error bars show s.d. based on three replicate infection experiments.

Figure 6

Diversity and environmental distribution of ‘Candidatus Nucleicultrix amoebiphila'. Phylogenetic trees based on amplicon sequences (>300 nucleotides length, >95% sequence similarity to the Nucleicultrix 16S rRNA) are shown. As 16S rRNA sequences from amplicon-based studies generally cover only short regions of the full-length gene, separate trees comprising sequences spanning variable regions V1–V2, V4–V6 and V7–V8, respectively, were calculated. Each tree depicts a subset of representative sequences retrieved from public databases. With all three data sets, Nucleicultrix-related sequences group into two distinct clades (marked yellow and green in the outer circle). Colors in the inner circle represent the environmental origin of the sequences; triangles indicate full-length sequences; the position of Nucleicultrix is labeled with an asterisk. Bars indicate an estimated evolutionary distance of 0.01.