The mechanisms of cell invasion in cnidarian-dinoflagellate symbiosis: Learning from parasitic strategies

Cnidarians such as corals, anemones and hydroids commonly form an intracellular symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium. Dinoflagellate symbionts are most often obtained anew from the environment during larval development, and, once acquired reside inside host-derived vacuoles within the cnidarian gastrodermal cells. In order to gain entry to host cells, the symbionts likely interact with innate immune receptors in the extracellular matrix, the first line of defense against microbial attack. While several innate immune pathways have been described in cnidarians, little is known about the specific receptor-ligand interactions that allow the symbiont to gain entry to host cells. Furthermore, it is unclear how these pathways are involved in enabling friendly microbes to reside within host cells while maintaining an immune response to harmful pathogens.  The invasion strategies of vertebrate intracellular parasites are well studied, especially those used by members of the Apicomplexa. Apicomplexan parasites have evolved mechanisms to evade immune receptors in the extracellular matrix and exploit specific receptors to their own benefit, to gain entry to host cells. Apicomplexans are closely related to dinoflagellates, both belonging to the infrakingdom Alveolata. The malaria parasite Plasmodium spp. has evolved the thromobospondin-related anonymous protein, or TRAP, that uses a thrombospondin structural homology repeat (TSR) domain to bind to a scavenger receptor (SRB1) on the hepatocyte cell surface and gain entry to the cell. This is of particular interest, as class B scavenger receptors are upregulated in the symbiotic state of two anemone species. The aims of the research presented in this thesis were to: (1) characterize the scavenger receptor (SR) repertoire in cnidarians; (2) characterize the TSR-domain-containing protein repertoire of cnidarians and their symbiotic dinoflagellates; and (3) establish, through experimental manipulation, the potential role for SR-TSR domain interactions at the onset of symbiosis in the sea anemone Aiptasia sp, a model system for the study of the cnidariandinoflagellate symbiosis.  In Chapter 2, I characterized the large and diverse SR repertoire of six cnidarian species. Cnidarians lack the classic SR type-A collagen domain-containing proteins that are common in humans, however the cnidarian SR cysteine-rich domain-containing protein repertoire is expanded and diverse. Phylogenetic analysis of SR type-B proteins defines two or three distinct groups. Functional experimental data presented here show that blocking SR binding sites with fucoidan significantly reduces dinoflagellate uptake by the anemone Aiptasia sp. These data provide further evidence that SRs are important to symbiont recognition and uptake, and may be an essential component of symbiont acquisition.  In Chapter 3, I investigated a SR ligand, the thrombospondin structural homology repeat, or TSR domain. In particular, I characterized the TSR-domain-containing protein repertoire of six cnidarian species and compared these proteins to vertebrate TSR proteins of known function. Searches revealed a large repertoire of TSR-domaincontaining proteins. Of particular interest is the large number of Adams metalloprotease-like proteins, a group that is common in both humans and cnidarians, suggesting that this is an ancestral TSR protein group. Phylogenetic analysis of TSR domains shows that binding motifs and 3-D folding sites are highly conserved. These data suggest that TSR domains are ancient and have changed very little in amino acid sequence from lower metazoans to vertebrates.  In Chapter 4, I explored the role of TSR-domain-containing proteins at the onset of symbiosis in the model Aiptasia sp. system. In functional experiments, aposymbiotic anemones were challenged with proteins and antibodies to either block or stimulate TSR domain binding. Symbiont uptake was measured over several time-points to determine the effects on symbiont acquisition. Adding an excess of TSR-domaincontaining protein or TSR synthetic peptide increased symbiont uptake, while blocking TSR domains prevented symbiont uptake. Finally, the addition of exogenous TGFβ to TSR antibody-challenged anemones, reversed the blocking effect. These data suggest that the immune-suppressive TGFβ pathway is involved in early onset of the symbiosis.  Since the TSR domain is implicated in the TGFβ pathway, these results support previous findings of the involvement of TGFβ in promoting tolerance of symbionts within the host. Apicomplexan parasites exploit scavenger receptor-TSR domain-binding to gain entry, and also use immune modulation to persist inside host cells. Data presented here suggest that dinoflagellates are utilizing the same mechanisms to form a mutualistic relationship with the cnidarian host.  Overall, the work presented here provides new information about several cnidarian extracellular matrix proteins, with searches revealing large repertoires of both scavenger receptors and TSR-domain-containing proteins. Functional data suggest that both protein families are involved in the cnidarian-dinoflagellate symbiosis. Searches of the dinoflagellate genome did not find a clear dinoflagellate homologue to the apicomplexan TRAP proteins. However, this research provides further evidence that similar receptor-ligand interactions are involved in the entry of both beneficial and pathogenic microbes to host cells. These results add to growing knowledge about the complex molecular pathways that enable and support cnidarian-dinoflagellate symbiosis. An understanding of the mechanisms that support healthy symbiosis is essential when trying to predict the vitality and productivity of reef ecosystems in the face of climate change.