The genome of an endosymbiotic methanogen is very similar to those of its free-living relatives.

The methanogenic endosymbionts of anaerobic protists represent the only known intracellular archaea, yet, almost nothing is known about genome structure and content in these lineages. Here, an almost complete genome of an intracellular Methanobacterium species was assembled from a metagenome derived from its host ciliate, a Heterometopus species. Phylogenomic analysis showed that the endosymbiont was closely related to free-living Methanobacterium isolates, and when compared with the genomes of free-living Methanobacterium, the endosymbiont did not show significant reduction in genome size or GC content. Additionally, the Methanobacterium endosymbiont genome shared the majority of its genes with its closest relative, though it did also contain unique genes possibly involved in interactions with the host via membrane-associated proteins, the removal of toxic by-products from host metabolism and the production of small signalling molecules. Though anaerobic ciliates have been shown to transmit their endosymbionts to daughter cells during division, the results presented here could suggest that the endosymbiotic Methanobacterium did not experience significant genetic isolation or drift and/or that this lineage was only recently acquired. Altogether, comparative genomic analysis identified genes potentially involved in the establishment and maintenance of the symbiosis, as well provided insight into the genomic consequences for an intracellular archaeum.

Insights into the metabolic functioning of a multipartner ciliate symbiosis from oxygen-depleted sediments.

Symbioses between anaerobic or microaerophilic protists and prokaryotes are common in anoxic and oxygen-depleted habitats ranging from marine sediments to gastrointestinal tracts. Nevertheless, little is known about the mechanisms of metabolic interaction between partners. In these putatively syntrophic associations, consumption of fermentative end products (e.g., hydrogen) by the prokaryotic symbionts is thought to facilitate protistan anaerobic metabolism. Here, we employed metagenomic and metatranscriptomic sequencing of a microaerophilic or anaerobic karyorelictid ciliate and its prokaryotic symbionts from oxygen-depleted Santa Barbara Basin (CA, USA) sediments to assess metabolic coupling within this consortium. This sequencing confirmed the predominance of deltaproteobacterial symbionts from the Families Desulfobacteraceae and Desulfobulbaceae and suggested active symbiont reduction of host-provided sulphate, transfer of small organic molecules from host to symbionts and hydrogen cycling among the symbionts. In addition, patterns of gene expression indicated active cell division by the symbionts, their growth via autotrophic processes and nitrogen exchange with the ciliate host. Altogether, this research underscores the importance of symbiont metabolism to host fermentative metabolism and, thus, likely its success in anoxic and low-oxygen habitats, but also suggests ciliate-associated prokaryotes play a role in important biogeochemical processes.

The uptake and excretion of partially oxidized sulfur expands the repertoire of energy resources metabolized by hydrothermal vent symbioses.

Symbiotic associations between animals and chemoautotrophic bacteria crowd around hydrothermal vents. In these associations, symbiotic bacteria use chemical reductants from venting fluid for the energy to support autotrophy, providing primary nutrition for the host. At vents along the Eastern Lau Spreading Center, the partially oxidized sulfur compounds (POSCs) thiosulfate and polysulfide have been detected in and around animal communities but away from venting fluid. The use of POSCs for autotrophy, as an alternative to the chemical substrates in venting fluid, could mitigate competition in these communities. To determine whether ESLC symbioses could use thiosulfate to support carbon fixation or produce POSCs during sulfide oxidation, we used high-pressure, flow-through incubations to assess the productivity of three symbiotic mollusc genera-the snails Alviniconcha spp. and Ifremeria nautilei, and the mussel Bathymodiolus brevior-when oxidizing sulfide and thiosulfate. Via the incorporation of isotopically labelled inorganic carbon, we found that the symbionts of all three genera supported autotrophy while oxidizing both sulfide and thiosulfate, though at different rates. Additionally, by concurrently measuring their effect on sulfur compounds in the aquaria with voltammetric microelectrodes, we showed that these symbioses excreted POSCs under highly sulfidic conditions, illustrating that these symbioses could represent a source for POSCs in their habitat. Furthermore, we revealed spatial disparity in the rates of carbon fixation among the animals in our incubations, which might have implications for the variability of productivity in situ. Together, these results re-shape our thinking about sulfur cycling and productivity by vent symbioses, demonstrating that thiosulfate may be an ecologically important energy source for vent symbioses and that they also likely impact the local geochemical regime through the excretion of POSCs.

Intracellular Oceanospirillales inhabit the gills of the hydrothermal vent snail Alviniconcha with chemosynthetic, γ-Proteobacterial symbionts.

Associations between bacteria from the γ-Proteobacterial order Oceanospirillales and marine invertebrates are quite common. Members of the Oceanospirillales exhibit a diversity of interactions with their various hosts, ranging from the catabolism of complex compounds that benefit host growth to attacking and bursting host nuclei. Here, we describe the association between a novel Oceanospirillales phylotype and the hydrothermal vent snail Alviniconcha. Alviniconcha typically harbour chemoautotrophic γ- or ε-Proteobacterial symbionts inside their gill cells. Via fluorescence in situ hybridization and transmission electron microscopy, we observed an Oceanospirillales phylotype (named AOP for 'Alviniconcha Oceanospirillales phylotype') in membrane-bound vacuoles that were separate from the known γ- or ε-Proteobacterial symbionts. Using quantitative polymerase chain reaction, we surveyed 181 Alviniconcha hosting γ-Proteobacterial symbionts and 102 hosting ε-Proteobacterial symbionts, and found that the population size of AOP was always minor relative to the canonical symbionts (median 0.53% of the total quantified 16S rRNA genes). Additionally, we detected AOP more frequently in Alviniconcha hosting γ-Proteobacterial symbionts than in those hosting ε-Proteobacterial symbionts (96% and 5% of individuals respectively). The high incidence of AOP in γ-Proteobacteria hosting Alviniconcha implies that it could play a significant ecological role either as a host parasite or as an additional symbiont with unknown physiological capacities.

Metatranscriptomics reveal differences in in situ energy and nitrogen metabolism among hydrothermal vent snail symbionts.

Despite the ubiquity of chemoautotrophic symbioses at hydrothermal vents, our understanding of the influence of environmental chemistry on symbiont metabolism is limited. Transcriptomic analyses are useful for linking physiological poise to environmental conditions, but recovering samples from the deep sea is challenging, as the long recovery times can change expression profiles before preservation. Here, we present a novel, in situ RNA sampling and preservation device, which we used to compare the symbiont metatranscriptomes associated with Alviniconcha, a genus of vent snail, in which specific host-symbiont combinations are predictably distributed across a regional geochemical gradient. Metatranscriptomes of these symbionts reveal key differences in energy and nitrogen metabolism relating to both environmental chemistry (that is, the relative expression of genes) and symbiont phylogeny (that is, the specific pathways employed). Unexpectedly, dramatic differences in expression of transposases and flagellar genes suggest that different symbiont types may also have distinct life histories. These data further our understanding of these symbionts' metabolic capabilities and their expression in situ, and suggest an important role for symbionts in mediating their hosts' interaction with regional-scale differences in geochemistry.

Induction of oral tolerance towards hepatitis B envelope antigens in a murine model.

BACKGROUND: Hepatitis B virus (HBV) is a non-cytopathic virus, and the hepatocellular injury that occurs as a consequence of HBV infection is mediated by the host antiviral immune response. Subjects with natural tolerance to HBV have minimal or no liver injury despite chronic viremia. We have shown that immune tolerance towards viruses can be induced by oral administration of viral proteins. AIMS: To test whether oral induction of tolerance can be induced towards HBV antigens, and whether oral tolerance induction downregulates preexisting anti-HBV immune response. METHODS: Oral tolerance was induced via feeding of five low oral doses of HBV proteins (HBsAg+preS1+preS2, BioHepB). This was followed by two inoculations with the BioHepB vaccine. Humoral immune tolerance was evaluated by measuring serum levels of anti-HBs antibody titers at monthly intervals. To determine if oral tolerance induction downregulates pre-existing anti-HBs immunity, mice were inoculated twice with the BioHepB vaccine, followed by feeding of BioHepB-HBV proteins. RESULTS: Feeding of HBV proteins markedly inhibited production of anti-HBs antibodies in naive mice. Anti-HBs titers were 45 versus 135 mIU/ml, in tolerized versus non-tolerized controls (P<0.005). Moreover, oral tolerance induction effectively down-regulated pre-existing immunity and reduced the anti-HBs titers in previously immunized mice to 112 versus 223 mIU/ml, in tolerized compared with non-tolerized controls (P<0.01). CONCLUSIONS: Induction of oral tolerance towards HBV proteins downregulates the antiviral humoral immune response in naive mice, and in the presence of preexisting anti-HBV immunity. This approach should be further investigated as a method for alleviation of antiviral-mediated liver injury in chronic HBV hepatitis.

Induction of oral tolerance in splenocyte recipients toward pretransplant antigens ameliorates chronic graft versus host disease in a murine model.

Chronic graft versus host disease (cGVHD) is a major complication that can develop after bone marrow transplantation. It involves an immune-mediated attack by transplanted donor lymphocytes, and often results in inflammatory damage of host target organs. Immune hyporesponsiveness induced by oral antigen administration has been recently shown to prevent the development of cGVHD in a murine model. The aim of this study was to evaluate whether tolerance induction in bone marrow transplant (BMT) recipients after transplantation, toward their pretransplant antigens, can alleviate preexisting cGVHD in a mouse model. cGVHD was generated by infusing 2.5 x 10(7) splenocytes from B10.D2 donor mice, to sublethally irradiated (6 Gy) BALB/c recipient mice, which differ by minor histocompatibility antigens. Transplantation resulted in cGVHD, with characteristic scleroderma-like cutaneous fibrosis, increased skin collagen content, decreased body weight, and hepatic and small bowel inflammation. Oral tolerance was induced by feeding recipient BALB/c mice with proteins extracted from BALB/c splenocytes for 11 days after B10.D2 splenocyte transplantation. Tolerance induction was evidenced by a significant reduction in mixed lymphocyte response of effector splenocytes from tolerant BALB/c mice transplanted with B10.D2 splenocytes against BALB/c target splenocytes. Oral tolerance decreased skin collagen deposits. Reduction of collagen alpha1(I) gene expression and skin collagen were shown by in situ hybridization and histochemistry, respectively. Liver and bowel biopsy specimens revealed less inflammation. Serum IL-10 levels were higher in tolerant mice than in controls, whereas IFNgamma was significantly reduced. Oral tolerance of BMT recipients toward their pretransplant antigens after splenocyte transplantation down-regulated the immune attack by transplanted cells, thus ameliorating cGVHD.