Starvation differentially affects gene expression, immunity and pathogen susceptibility across symbiotic states in a model cnidarian.

Mutualistic symbioses between cnidarians and photosynthetic algae are modulated by complex interactions between host immunity and environmental conditions. Here, we investigate how symbiosis interacts with food limitation to influence gene expression and stress response programming in the sea anemone Exaiptasia pallida (Aiptasia). Transcriptomic responses to starvation were similar between symbiotic and aposymbiotic Aiptasia; however, aposymbiotic anemone responses were stronger. Starved Aiptasia of both symbiotic states exhibited increased protein levels of immune-related transcription factor NF-κB, its associated gene pathways, and putative target genes. However, this starvation-induced increase in NF-κB correlated with increased immunity only in symbiotic anemones. Furthermore, starvation had opposite effects on Aiptasia susceptibility to pathogen and oxidative stress challenges, suggesting distinct energetic priorities under food scarce conditions. Finally, when we compared starvation responses in Aiptasia to those of a facultative coral and non-symbiotic anemone, 'defence' responses were similarly regulated in Aiptasia and the facultative coral, but not in the non-symbiotic anemone. This pattern suggests that capacity for symbiosis influences immune responses in cnidarians. In summary, expression of certain immune pathways-including NF-κB-does not necessarily predict susceptibility to pathogens, highlighting the complexities of cnidarian immunity and the influence of symbiosis under varying energetic demands.

Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor.

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

Molecular and Biochemical Approaches to Study the Evolution of NF-κB Signaling in Basal Metazoans.

Extensive genomic and transcriptomic sequencing over the past decade has revealed NF-κB signaling pathway homologs in organisms basal to insects, for example, in members of the phyla Cnidaria (e.g., sea anemones, corals, hydra, jellyfish) and Porifera (sponges), and in several single-celled protists (e.g., Capsaspora owczarzaki, some choanoflagellates). Therefore, methods are required to study the function of NF-κB and its pathway members in early branching organisms, many of which do not have histories as model organisms. Here, we describe a combination of cellular, molecular, and biochemical techniques that have been used for studying NF-κB, and related pathway proteins, in some of these basal organisms. These methods are useful for studying the evolution of NF-κB signaling, and may be adaptable to the study of NF-κB in other non-model organisms.