Transmission, Maintenance and Cell cycle in the Endoriftia Symbiosis

The sulfide-oxidizing bacteria Candidatus Endoriftia persephone live as endosymbionts in Riftia pachyptila, the giant tubeworm from the deep-sea hydrothermal vents of the East Pacific. Each host generation has to acquire the specific symbionts anew from the environment. This horizontal symbiont transmission requires special recognition and uptake mechanisms ensuring continuity. After infection of the host, the symbionts migrate through different layers of host tissue until they become engulfed by vacuoles within bacteriocyte host cells, which form a new organ termed the trophosome. The infection process and the living together of the two partners are orchestrated by yet unknown mechanisms. It is unclear to which extent the symbionts and/or the host regulate the molecular processes necessary for uptake, migration, maintenance and cell cycle within the bacteriocyte host cells. In this study these processes will be investigated using immuno-histochemistry and immuno-ultrahistochemistry with custom monospecific antibodies against selected effector proteins of the symbionts. Mining the published metagenome and functional proteome data of Endoriftia I will select approximately 20 target genes/proteins involved in essential symbiont - host interactions. The following host stages will be used to search for expressed genes: the larvae in metamorphosis; the small juveniles with developing trophosome; the juveniles with one-lobule trophosome and the adult tubeworms with multi-lobule trophosome. So far, free-living symbionts have been found in the surrounding vent environment, in the tube and in mucus attached to the metamorphosing larvae. They appear as dividing rods and small cocci, whereas during migration through the host`s tissue and at the start of the trophosome development the symbionts are dividing rods only. During the cell cycle with terminal differentiation, the symbionts develop from dividing rods to dividing small cocci to non-dividing large cocci and finally become degraded in the peripheral zone of the trophosome. Thus I will follow the expression patterns of selected symbiont genes with refined spatial and temporal resolution. In detail, I will concentrate on the symbiont proteins involved in mobility, important to reach the host and to migrate through host tissue, in chemotaxis and adhesion, essential to find and attach to the host, in the immune response to counteract the host immune system, and in cell cycle proteins to regulate DNA synthesis and division. Uncovering the involved proteins will enable us to better understand which mechanisms this symbiont uses to find and enter the host, to migrate through host tissues, and to establish itself as an endosymbiont in an organ. It will not only add to our understanding of the Riftia model system specifically, and the manifold diversity of microbial symbioses in general, but also will help us to understand common molecular pathways of beneficial and pathogen bacteria, thus cross linking the disciplines of biology and medicine.

The cell cycles of the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus En- doriftia persephone are tightly interconnected. The symbionts appear in different morphology- dividing rods and small cocci in the inner and middle zone, non-dividing large cocci and degrading cocci in the peripheral zone of the trophosome - the symbiont-housing organ. At some point during the life cycle the symbionts stop to divide but grow larger. An equilibrium of host and symbiont cells is maintained in a way which allows for rapid growth, while the terminal differentiation of host cells leads eventually to apoptosis and autophagy during which the symbionts are degraded. In this project, we showed that the symbionts are able to maintain their cell cycle and to survive even when the host starts to deteriorate. Thus, the symbionts are potentially able to leave the dying host and inoculate the free-living community.We measured the expression level of selected cell cycle proteins. We compared the expression levels of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and the cell cycle proteins of the symbionts in hosts with different conditions indicated by the color of the trophosome. When R. pachyptila cannot access sufficient sulfide, the trophosome turns black and the symbionts seem to be chemoautotrophically less active as the reduced expression of RuBisCo implies. As expected, the symbionts of the green trophosome, with its high content of elemental sulfur, had a high level of RuBisCo, but surprisingly, the expression level of Ru- BisCo of the pink trophosome, which was described as dying/deteriorating was not significantly different from the green. Furthermore, the expression pattern of the cell cycle proteins was also not significantly different, meaning, that the deteriorating pink trophosome houses viable symbionts, which can leave the dying host, attach to surfaces, start to divide, and thus inoculate the free-living symbiont population. Immunohistochemically determined spatial distribution within a trophosome lobule showed that the chromosome partitioning protein ParA accumulated in the symbionts of the outer zones of the trophosome. This overexpression of ParA possibly leads to activation of the SOS protein SulA and further to cell division arrest of the symbiont. However, the effector proteins or pathways of the host, which lead to the cell division arrest in the symbionts, still need to be determined.