Role of host ciliate Paramecium bursaria mitochondria and trichocysts for symbiotic Chlorella variabilis attachment beneath the host cell cortex.

Symbiotic Chlorella variabilis is encased in the perialgal vacuole (PV) membrane of ciliate Paramecium bursaria. The PV membrane is stably anchored below the host cell cortex by adhesion to host mitochondria. Host trichocysts, which are defensive organelles against predators, are present in the mitochondria and PV membrane vicinity. The mechanism by which PV attaches beneath the host cell cortex remains unknown. When P. bursaria is centrifuged at high speed, the symbiotic algae are displaced from the host cell cortex and concentrate at the posterior end. When centrifugation is stopped, the dislocated algae reattach beneath the host cell cortex with fast cytoplasmic streaming. The densities of mitochondria and trichocysts before and after centrifugation were compared using indirect immunofluorescence microscopy with monoclonal antibodies. Almost all trichocysts were shed by high-speed centrifugation, but dislocated algae could reattach even in the absence of trichocysts. In contrast, host mitochondria were unaffected in localization and number, and the dislocated algae also reattached. These findings suggest trichocysts are unnecessary for algal relocalization and that mitochondria are colocalized with the algae. However, many mitochondria were also present in the cell's anterior region without symbiotic algae. Therefore, not all areas with mitochondria contained algae, but there was an algal localization bias within the host cell.

A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum.

The Gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. It is known that an infection of this bacterium induces high level expressions of the host hsp60 and hsp70 genes, and the host cell acquires both heat-shock and high salt resistances. In addition, an infectious form of H. obtusa-specific 63-kDa periplasmic protein with a DNA-binding domain in its amino acid sequence is secreted into the host macronucleus after invasion into the macronucleus and remain within the nucleus. These facts suggest that binding of the 63-kDa protein to the host macronuclear DNA causes changes in the host gene expressions and enhances an environmental adaptability of the host cells. This 63-kDa protein was renamed as periplasmic region protein 1 (PRP1) to distinguish it from other proteins with similar molecular weights. To confirm whether PRP1 indeed binds to the host DNA, SDS-DNA PAGE and DNA affinity chromatography with calf thymus DNA and P. caudatum DNA were conducted and confirmed that PRP1 binds weakly to the P. caudatum DNA with a monoclonal antibody raised for the 63-kDa protein.

Endosymbiotic Chlorella variabilis reduces mitochondrial number in the ciliate Paramecium bursaria.

Extant symbioses illustrate endosymbiosis is a driving force for evolution and diversification. In the ciliate Paramecium bursaria, the endosymbiotic alga Chlorella variabilis in perialgal vacuole localizes beneath the host cell cortex by adhesion between the perialgal vacuole membrane and host mitochondria. We investigated whether host mitochondria are also affected by algal endosymbiosis. Transmission electron microscopy of host cells showed fewer mitochondria beneath the algae-bearing host cell cortex than that of alga-free cells. To compare the density and distribution of host mitochondria with or without symbiotic algae, we developed a monoclonal antibody against Paramecium mitochondria. Immunofluorescence microscopy with the monoclonal antibody showed that the mitochondrial density of the algae-bearing P. bursaria was significantly lower than that of the alga-free cells. The total cell protein concentration of alga-free P. bursaria cells was approximately 1.8-fold higher than that of algae-bearing cells, and the protein content of mitochondria was significantly higher in alga-free cells than that in the algae-bearing cells. These results corresponded with those obtained by transmission electron and immunofluorescence microscopies. This paper shows that endosymbiotic algae affect reduced mitochondrial number in the host P. bursaria significantly.

Mechanisms for establishing primary and secondary endosymbiosis in Paramecium.

Primary (eukaryote and procaryote) and secondary (eukaryote and eukaryote) endosymbioses are driving forces in eukaryotic cell evolution. These phenomena are still contributing to acquire new cell structures and functions. To understand mechanisms for establishment of each endosymbiosis, experiments that can induce endosymbiosis synchronously by mixing symbionts isolated from symbiont-bearing host cells and symbiont-free host cells are indispensable. Recent progress on endosymbiosis using Paramecium and their endonuclear symbiotic bacteria Holospora or symbiotic green alga Chlorella has been remarkable, providing excellent opportunities for elucidating host-symbiont interactions. These organisms are now becoming model organisms to know the mechanisms for establishing primary and secondary endosymbioses. Based on experiments of many researchers, we introduce how these endosymbionts escape from the host lysosomal fusion, how they migrate in the host cytoplasm to localize specific locations within the host, how their species specificity and strain specificity of the host cells are controlled, how their life cycles are controlled, how they escape from the host cell to infect more young host cell, how they affect the host viability and gene expression, what kind of substances are needed in these phenomena, and what changes had been induced in the symbiont and the host genomes.

Peculiar Paramecium Hosts Fail to Establish a Stable Intracellular Relationship With Legionella pneumophila.

Legionella pneumophila, an intracellular human pathogen, establishes intracellular relationships with several protist hosts, including Paramecium caudatum. L. pneumophila can escape the normal digestion process and establish intracellular relationships in Paramecium. In this study, we identify new Paramecium strains that significantly reduce the number of L. pneumophila during infection. As a result, stable intracellular relationships between L. pneumophila and these Paramecium strains were not observed. These digestion-type Paramecium also showed high efficiency for Escherichia coli elimination compared to other strains of Paramecium. These results suggest that the digestion-type strains identified have high non-specific digestion activity. Although we evaluated the maturation process of Legionella-containing vacuoles (LCVs) in the Paramecium strains using LysoTracker, there were no discriminative changes in these LCVs compared to other Paramecium strains. Detailed understanding of the mechanisms of high digestion efficiency in these strains could be applied to water purification technologies and L. pneumophila elimination from environmental water.

Genome plasticity in Paramecium bursaria revealed by population genomics.

BACKGROUND: Ciliates are an ancient and diverse eukaryotic group found in various environments. A unique feature of ciliates is their nuclear dimorphism, by which two types of nuclei, the diploid germline micronucleus (MIC) and polyploidy somatic macronucleus (MAC), are present in the same cytoplasm and serve different functions. During each sexual cycle, ciliates develop a new macronucleus in which newly fused genomes are extensively rearranged to generate functional minichromosomes. Interestingly, each ciliate species seems to have its way of processing genomes, providing a diversity of resources for studying genome plasticity and its regulation. Here, we sequenced and analyzed the macronuclear genome of different strains of Paramecium bursaria, a highly divergent species of the genus Paramecium which can stably establish endosymbioses with green algae. RESULTS: We assembled a high-quality macronuclear genome of P. bursaria and further refined genome annotation by comparing population genomic data. We identified several species-specific expansions in protein families and gene lineages that are potentially associated with endosymbiosis. Moreover, we observed an intensive chromosome breakage pattern that occurred during or shortly after sexual reproduction and contributed to highly variable gene dosage throughout the genome. However, patterns of copy number variation were highly correlated among genetically divergent strains, suggesting that copy number is adjusted by some regulatory mechanisms or natural selection. Further analysis showed that genes with low copy number variation among populations tended to function in basic cellular pathways, whereas highly variable genes were enriched in environmental response pathways. CONCLUSIONS: We report programmed DNA rearrangements in the P. bursaria macronuclear genome that allow cells to adjust gene copy number globally according to individual gene functions. Our results suggest that large-scale gene copy number variation may represent an ancient mechanism for cells to adapt to different environments.

Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites.

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

Identification of novel Legionella genes required for endosymbiosis in Paramecium based on comparative genome analysis with Holospora spp.

The relationship between Legionella and protist hosts has a huge impact when considering the infectious risk in humans because it facilitates the long-term replication and survival of Legionella in the environment. The ciliate Paramecium is considered to be a protist host for Legionella in natural environments, but the details of their endosymbiosis are largely unknown. In this study, we determined candidate Legionella pneumophila genes that are likely to be involved in the establishment of endosymbiosis in Paramecium caudatum by comparing the genomes of Legionella spp. and Holospora spp. that are obligate endosymbiotic bacteria in Paramecium spp. Among the candidate genes, each single deletion mutant for five genes (lpg0492, lpg0522, lpg0523, lpg2141 and lpg2398) failed to establish endosymbiosis in P. caudatum despite showing intracellular growth in human macrophages. The mutants exhibited no characteristic changes in terms of their morphology, multiplication rate or capacity for modulating the phagosomes in which they were contained, but their resistance to lysozyme decreased significantly. This study provides insights into novel factors required by L. pneumophila for endosymbiosis in P. caudatum, and suggests that endosymbiotic organisms within conspecific hosts may have shared genes related to effective endosymbiosis establishment.

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires' disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries' disease, understanding the ecology of L. pneumophila in the environment is essential.

High degree of specificity in the association between symbiotic betaproteobacteria and the host Euplotes (Ciliophora, Euplotia).

The Betaproteobacteria-Euplotes association is an obligatory symbiotic system involving a monophyletic group of ciliate species and two betaproteobacteria species which can be alternatively present. Recent data showed that this relationship has been established more than once and that several symbiont-substitution events took place, revealing a complex and intriguing evolutionary path. Due to the different evolutionary pathways followed by the different symbionts, each bacterial strain could have differentially evolved and/or lost functional traits. Therefore, we performed re-infection experiments, both by phagocytosis and by microinjection, to test the possible functional role of the different bacteria towards the ciliates. Our results confirm that the growth capacity of the host is indissolubly linked to the presence of its original symbionts. Results of the attempts of re-infection by phagocytosis showed that none of the bacteria is able to successfully colonize the host cytoplasm in this way, even if regularly ingested. Re-infection by microinjection succeed only in one case. Such results point to a high degree of specificity in the interactions between bacteria and Euplotes even after the invasion step. Due to a co-evolutive pathway of reciprocal adaptation, different degree of re-colonization ability could have been conserved by the different species and strains of the symbionts.

Ciliate Paramecium is a natural reservoir of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment.

Analysis of amino acid and codon usage in Paramecium bursaria.

The ciliate Paramecium bursaria harbors the green-alga Chlorella symbionts. We reassembled the P. bursaria transcriptome to minimize falsely fused transcripts, and investigated amino acid and codon usage using the transcriptome data. Surface proteins preferentially use smaller amino acid residues like cysteine. Unusual synonymous codon and amino acid usage in highly expressed genes can reflect a balance between translational selection and other factors. A correlation of gene expression level with synonymous codon or amino acid usage is emphasized in genes down-regulated in symbiont-bearing cells compared to symbiont-free cells. Our results imply that the selection is associated with P. bursaria-Chlorella symbiosis.

Symbiotic Chlorella variabilis incubated under constant dark conditions for 24 hours loses the ability to avoid digestion by host lysosomal enzymes in digestive vacuoles of host ciliate Paramecium bursaria.

Endosymbiosis between symbiotic Chlorella and alga-free Paramecium bursaria cells can be induced by mixing them. To establish the endosymbiosis, algae must acquire temporary resistance to the host lysosomal enzymes in the digestive vacuoles (DVs). When symbiotic algae isolated from the alga-bearing paramecia are kept under a constant dark conditions for 24 h before mixing with the alga-free paramecia, almost all algae are digested in the host DVs. To examine the cause of algal acquisition to the host lysosomal enzymes, the isolated algae were kept under a constant light conditions with or without a photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea for 24 h, and were mixed with alga-free paramecia. Unexpectedly, most of the algae were not digested in the DVs irrespective of the presence of the inhibitor. Addition of 1 mM maltose, a main photosynthetic product of the symbiotic algae or of a supernatant of the isolated algae kept for 24 h under a constant light conditions, did not rescue the algal digestion in the DVs. These observations reveal that unknown factors induced by light are a prerequisite for algal resistance to the host lysosomal enzymes.

Draft genome sequences of three Holospora species (Holospora obtusa, Holospora undulata, and Holospora elegans), endonuclear symbiotic bacteria of the ciliate Paramecium caudatum.

We present draft genome sequences of three Holospora species, hosted by the ciliate Paramecium caudatum; that is, the macronucleus-specific H. obtusa and the micronucleus-specific H. undulata and H. elegans. We investigate functions of orthologous core genes conserved across the three Holospora species, which may be essential for the infection and survival in the host nucleus.

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

BACKGROUND: The 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. RESULTS: To 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 70 kDa protein and glutathione S-transferase. CONCLUSIONS: This 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.

Draft Genome Sequence of Holospora undulata Strain HU1, a Micronucleus-Specific Symbiont of the Ciliate Paramecium caudatum.

Holospora undulata is a micronucleus-specific symbiont of the ciliate Paramecium caudatum. We report here the draft genome sequence of H. undulata strain HU1. This genome information will contribute to the study of symbiosis between H. undulata and the host P. caudatum.

Synchronous induction of detachment and reattachment of symbiotic Chlorella spp. from the cell cortex of the host Paramecium bursaria.

Paramecium bursaria harbor several hundred symbiotic Chlorella spp. Each alga is enclosed in a perialgal vacuole membrane, which can attach to the host cell cortex. How the perialgal vacuole attaches beneath the host cell cortex remains unknown. High-speed centrifugation (> 1000×g) for 1min induces rapid detachment of the algae from the host cell cortex and concentrates the algae to the posterior half of the host cell. Simultaneously, most of the host acidosomes and lysosomes accumulate in the anterior half of the host cell. Both the detached algae and the dislocated acidic vesicles recover their original positions by host cyclosis within 10min after centrifugation. These recoveries were inhibited if the host cytoplasmic streaming was arrested by nocodazole. Endosymbiotic algae during the early reinfection process also show the capability of desorption after centrifugation. These results demonstrate that adhesion of the perialgal vacuole beneath the host cell cortex is repeatedly inducible, and that host cytoplasmic streaming facilitates recovery of the algal attachment. This study is the first report to illuminate the mechanism of the induction to desorb for symbiotic algae and acidic vesicles, and will contribute to the understanding of the mechanism of algal and organelle arrangements in Paramecium.

Cell division and density of symbiotic Chlorella variabilis of the ciliate Paramecium bursaria is controlled by the host's nutritional conditions during early infection process.

The association of ciliate Paramecium bursaria with symbiotic Chlorella sp. is a mutualistic symbiosis. However, both the alga-free paramecia and symbiotic algae can still grow independently and can be reinfected experimentally by mixing them. Effects of the host's nutritional conditions against the symbiotic algal cell division and density were examined during early reinfection. Transmission electron microscopy revealed that algal cell division starts 24 h after mixing with alga-free P. bursaria, and that the algal mother cell wall is discarded from the perialgal vacuole membrane, which encloses symbiotic alga. Labelling of the mother cell wall with Calcofluor White Stain, a cell-wall-specific fluorochrome, was used to show whether alga had divided or not. Pulse labelling of alga-free P. bursaria cells with Calcofluor White Stain-stained algae with or without food bacteria for P. bursaria revealed that the fluorescence of Calcofluor White Stain in P. bursaria with bacteria disappeared within 3 days after mixing, significantly faster than without bacteria. Similar results were obtained both under constant light and dark conditions. This report is the first describing that the cell division and density of symbiotic algae of P. bursaria are controlled by the host's nutritional conditions during early infection.

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria.

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20µm diameter did not induce budding of the DVs. Microbeads with 0.80µm diameter produced DVs of 5-10µm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5µm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00µm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding.

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 Holospora's 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.