Masahiro Fujishima

Endosymbionts in Paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holosporas invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary.Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis.

Symbiotic alga Chlorella vulgaris of the ciliate Paramecium bursaria shows temporary resistance to host lysosomal enzymes during the early infection process

Summary.Paramecium bursaria free of symbiotic Chlorella species can be experimentally reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. Isolated symbiotic algae were cloned, mixed with the algae-free P. bursaria at 25 ± 1 °C for 1.5 min, washed and chased, with or without fixation 3 h after mixing. Though genetically identical, a few of the algae were not digested but coexisted with the digested ones in the same vacuole after lysosomal fusion. Light microscopy showed that algal fate did not depend on cell cycle stage or location in the vacuole. Electron microscopy showed that the nondigested algae were not protected by a perialgal vacuole membrane in the digestive vacuole. Moreover, this phenomenon was also observed in the presence of cycloheximide and puromycin, which are known to inhibit algal and host protein synthesis, respectively. These observations suggest that a few algae can acquire temporary resistance to host lysosomal enzymes in order to establish endosymbiosis without algal protein synthesis.

Cycloheximide Induces Synchronous Swelling of Perialgal Vacuoles Enclosing Symbiotic Chlorella vulgaris and Digestion of the Algae in the Ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the hosts cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the hosts cell membrane, were condensed and stained with Gomoris solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.

Comparison of gene expression of Paramecium bursaria with and without Chlorella variabilis symbionts

BackgroundThe ciliate Paramecium bursaria harbors several hundred cells of the green-alga Chlorella sp. in their cytoplasm. Irrespective of the mutual relation between P. bursaria and the symbiotic algae, both cells retain the ability to grow without the partner. They can easily reestablish endosymbiosis when put in contact with each other. Consequently, P. bursaria is an excellent model for studying cell–cell interaction and the evolution of eukaryotic cells through secondary endosymbiosis between different protists. Despite the importance of this organism, no genomic resources have been identified for P. bursaria to date. This investigation compared gene expressions through RNA-Seq analysis and de novo transcriptome assembly of symbiont-free and symbiont-bearing host cells.ResultsTo expedite the process of gene discovery related to the endosymbiosis, we have undertaken Illumina deep sequencing of mRNAs prepared from symbiont-bearing and symbiont-free P. bursaria cells. We assembled the reads de novo to build the transcriptome. Sequencing using Illumina HiSeq2000 platform yielded 232.3 million paired-end sequence reads. Clean reads filtered from the raw reads were assembled into 68,175 contig sequences. Of these, 10,557 representative sequences were retained after removing Chlorella sequences and lowly expressed sequences. Nearly 90% of these transcript sequences were annotated by similarity search against protein databases. We identified differentially expressed genes in the symbiont-bearing P. bursaria cells relative to the symbiont-free cells, including heat shock 70xa0kDa protein and glutathione S-transferase.ConclusionsThis is the first reported comprehensive sequence resource of Paramecium – Chlorella endosymbiosis. Results provide some keys for the elucidation of secondary endosymbiosis in P. bursaria. We identified P. bursaria genes that are differentially expressed in symbiont-bearing and symbiont-free conditions.

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria.

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomoris staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomoris thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomoris staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.

Infection and Maintenance of Holospora Species in Paramecium caudatum

Infectious forms of Holospora species have a cytoplasmic region and a periplasmic lumen with an electron-translucent invasion tip. Bacterial 89-kDa proteins with two actin-binding motifs translocate from the invasion tip lumen to outside the tip when infectious forms are ingested in digestive vacuoles of a Paramecium cell. With the invasion tip, the bacteria disrupt the digestive vacuole membrane, appear in the host cytoplasm, migrate to their target nucleus, distinguish host nuclear envelopes of two kinds through affinity between the bacterial lipopolysaccharides of the outer membranes and an unknown substance of the target nuclear envelope, and then penetrate the target nuclear envelope with the invasion tip. The first bacterium invades the host nucleus within 10 min after mixing. After endosymbiosis the bacteria alter host gene expression, providing an excellent opportunity for elucidating host-symbiont interactions between eukaryotic and prokaryotic cells and establishment of new associations leading to eukaryotic cell evolution.

Secondary symbiosis between Paramecium and Chlorella cells.

Each symbiotic Chlorella species of Paramecium bursaria is enclosed in a perialgal vacuole (PV) membrane derived from the host digestive vacuole (DV) membrane. Algae-free paramecia and symbiotic algae are capable of growing independently and paramecia can be reinfected experimentally by mixing them. This phenomenon provides an excellent model for studying cell-to-cell interaction and the evolution of eukaryotic cells through secondary endosymbiosis between different protists. However, the detailed algal infection process remains unclear. Using pulse labeling of the algae-free paramecia with the isolated symbiotic algae and chase method, we found four necessary cytological events for establishing endosymbiosis. (1) At about 3 min after mixing, some algae show resistance to the host lysosomal enzymes in the DVs, even if the digested ones are present. (2) At about 30 min after mixing, the alga starts to escape from the DVs as the result of the budding of the DV membrane into the cytoplasm. (3) Within 15 min after the escape, the DV membrane enclosing a single green alga differentiates to the PV membrane, which provides protection from lysosomal fusion. (4) The alga localizes at the primary lysosome-less host cell surface by affinity of the PV to unknown structures of the host. At about 24 h after mixing, the alga multiplies by cell division and establishes endosymbiosis. Infection experiments with infection-capable and infection-incapable algae indicate that the infectivity of algae is based on their ability to localize beneath the host surface after escaping from the DVs. This algal infection process differs from known infection processes of other symbiotic or parasitic organisms to their hosts.

Localization of perialgal vacuoles beneath the host cell surface is not a prerequisite phenomenon for protection from the host's lysosomal fusion in the ciliate Paramecium bursaria.

In a ciliate Paramecium bursaria cell, each symbiotic 3-4-mum-diameter Chlorella cell is enclosed within a perialgal vacuole membrane. It localizes near trichocysts beneath the host cell surface. Gomoris staining of this surface shows that it is an acid phosphatase activity-negative area to 5-10mum depth. Trichocysts were removed by treatment with 1mg/ml lysozyme to elucidate whether algal protection from the host lysosomal fusion is controlled by localization of the perialgal vacuole membrane to the acid phosphatase activity-negative area or by the capability of the perialgal vacuole membrane to give protection from lysosomal fusion. The trichocyst-free cell reduced the acid phosphatase activity-negative area to less than 3mum depth at the dorsal surface. However, even though a part of the algal cell had been exposed in the acid phosphatase activity-positive area, the algae were able to attach beneath the host surface and to protect it from lysosomal fusion. Results of this study show that the perialgal vacuole membrane can give protection from host lysosomal fusion, and that the membrane does not require trichocysts for intracellular localization.

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25u2003°C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25u2003°C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.