Abstract
Lack of proper nutrition (malnutrition) or the complete absence of all food (starvation) have important consequences on the physiology of all organisms. In many cases, nutritional status affects immunity, but, for the most part, the relationship between nutrition and immunity has been limited to studies in vertebrates and terrestrial invertebrates. Herein, we describe a positive correlation between nutrition and immunity in the sea anemone Nematostella vectensis. Gene expression profiling of adult fed and starved anemones showed downregulation of many genes involved in nutrient metabolism and cellular respiration, as well as immune-related genes, in starved animals. Starved adult anemones also had reduced protein levels and DNA-binding activity of immunity-related transcription factor NF-κB. Starved juvenile anemones had increased sensitivity to bacterial infection and also had lower NF-κB protein levels, as compared to fed controls. Weighted Gene Correlation Network Analysis (WGCNA) revealed significantly correlated gene networks that were inversely associated with starvation. Based on the WGCNA and a reporter gene assay, we identified TRAF3 as a likely NF-κB target gene in N. vectensis. Overall, these experiments demonstrate a correlation between nutrition and immunity in a basal marine metazoan, and the results have implications for the survival of marine organisms as they encounter changing environments.
Significance Statement Adequate nutrition is required to sustain proper biological function. One factor threatening many marine organisms, as a result of modern day anthropogenic environmental changes, is nutrient availability. Here, we characterize transcriptional changes following food deprivation in the cnidarian model sea anemone Nematostella vectensis. We show that starvation is correlated with decreased expression of genes associated with nutrient metabolism and immunity, among others. Moreover, starvation reduces the level of expression and activity of immune regulatory transcription factor NF-κB and causes anemones to have increased susceptibility to bacterial infection. These results demonstrate that this basal organism responds at the transcriptional level to the absence of food, and that, in addition to changes in metabolic factors, starvation leads to a reduction in immunity.
Introduction
Maintenance of caloric needs is a requirement across the tree of life. Food scarcity is a challenge most heterotrophs encounter at various points during their lives – a situation that contributes to natural selection. The lack of proper nutrition (malnutrition) or the complete absence of food (starvation) has important consequences on an organism’s physiology. Starvation can lead to the slowing of metabolism1, and nutrition is one of the many factors that determine immune status of organisms2. Indeed, the adverse impact of poor nutrition on the immune system, including its inflammatory component, is well documented in vertebrates and some terrestrial invertebrates3-11. However, the interplay between nutrition and immunity has, for the most part, only been described in more derived organisms from insects to mammals.
The cnidarian model starlet sea anemone Nematostella vectensis (Nv) shows remarkable adaptability – being able to survive across a wide range of salinity, pH, and temperature – and it can be readily maintained and studied in the laboratory12. Nv also has exceptional regenerative abilities, being able to replace its entire oral end in ∼6 days following bisection13,14. Importantly, Nv can withstand periods of starvation for over a month, where its body size decreases in response to the lack of caloric intake while maintaining body proportionality15, suggesting that Nv can physiologically respond to changes in food availability. This ability is not limited to adult Nv, as previous work has shown that the timing of tentacle development in young Nv polyps is intertwined with feeding16. Nv is also distantly related to reef-building corals, where food availability has been shown to mitigate negative consequences of climate change17-19.
Proper resource allocation is vital for survival. Among energetic needs, the immune system is metabolically costly and subject to regulation20. Transcription factor NF-κB (nuclear factor kappa B) has been intensively studied in animals from insects to vertebrates for its involvement in development and immunity21. Although the basic NF-κB pathway in Nv has been characterized22 and has been linked to development23 and immunity24,25, the relationship between nutrition and immunity in Nv – or any cnidarian – remains vastly unexplored. Mammals have five different NF-κB proteins and flies have three, all of which are involved in some aspect of immunity21. Most basal organisms, including sponges and cnidarians, have single NF-κB proteins; however, the role of NF-κB in these organisms is less clear. Although NF-κB is regulated by post-translational mechanisms in organisms more derived than flies, there is evidence that NF-κB activity is regulated at the transcriptional level in many basal metazoans26-28. Examples of NF-κB target genes include cytokines and other immune response factors in mammals, and anti-microbial proteins such as cecropins in insects29-30. Furthermore, NF-κB signaling has been shown to play a key role in metabolic disease given its role in inflammation and the association of chronic inflammation with diseases such as obesity and diabetes6.
Herein, we have investigated the relationship between nutrition and immunity in Nv using gene expression profiling, characterization of transcription factor NF-κB, and susceptibility to infection with a bacterial pathogen. These results directly connect immunity and nutrition in a cnidarian, suggesting that nutritional status and immunity are evolutionarily linked processes.
Results
Genome-wide transcriptomic changes in N. vectensis in response to starvation
To assess the impact of reduced nutrition on N. vectensis (Nv), we performed genome-wide gene expression profiling using TaqSeq on eight clonal anemone pairs wherein single animals from clonal pairs were either fed routinely or starved for four weeks prior to RNA isolation (Fig. 1a). Clonal pairs were used because preliminary experiments showed heterogeneity in protein expression when comparing anemones of differing genetic backgrounds. To avoid positional effects after regeneration, we included animals regenerated from aboral and oral ends in both fed and starved groups (Extended Data Table 1). Raw sequencing reads from all 16 anemones ranged from 5.2 to 9.2 million. Alignment values for all samples ranged from 74.2 to 77.4%. DESeq2 identified 711 significant differentially expressed genes (DEGs) between starved and fed anemones while controlling for genetic background (FDR adjusted p-value < 0.1): 118 genes were upregulated and 593 genes were downregulated in starved relative to fed anemones (Fig. 1b). A full list of the differentially expressed genes is presented in Extended Data Dataset 1.
To assess overall transcriptional differences between fed and starved anemones, rlog- normalized gene expression data were used for Principal Components Analysis (PCA), which showed that nutritional status had a significant influence on gene expression (Fig. 1c, left; Adonis PERMANOVA ptreat = 0.02). Additionally, the effect of genet (i.e., genetic background) also had a significant impact on gene expression (Fig. 1c, Adonis PERMANOVA pgenet = 0.01). These differences in gene expression between fed and starved animals were also seen when comparing individual clonal pairs where each starved anemone showed a similar rightward shift along PC1 compared to its fed counterpart (Fig. 1c, right).
GO pathway response to starvation
To explore the functional response of Nv to starvation, a Gene Ontology (GO) enrichment analysis of “Biological Process” terms associated with significant DEGs (Extended Data Fig. 1) was performed using ranked p-values. Among genes showing positive log-fold changes following starvation, three prominent Biological Process trends emerged: RNA processing (i.e., RNA processing GO:0006396, RNA splicing GO:0008380, mRNA metabolic process GO:0016071), DNA processing (i.e., DNA metabolic process GO:0006259, DNA replication GO:0006260, DNA repair GO:0006281), and chromosome organization (i.e., chromatin remodeling GO:0006338, chromatin organization GO:0006325, covalent chromatin modification GO:0016569).
Consistent with results from the DEG analysis (Fig. 1b), more GO terms were underrepresented in starved anemones. Many of the underrepresented GO terms reflected the nutrient-deprived status of these animals, with terms related to nutrient metabolism (i.e., response to nutrient GO:0007584, lipid metabolic process GO:0006629, carbohydrate metabolic process GO:0005975, cellular amino acid metabolic process GO:0006520) and cellular respiration (i.e., electron transport chain GO:0022900, proton transmembrane transport GO:1902600) (Extended Data Fig. 1). Thus, these downregulated GO terms indicate that the anemones were indeed starved.
Of note, many immune-related terms were underrepresented following starvation (i.e., immune response GO:0006955, defense response GO:0006952, antigen processing and presentation GO:0019882), as well as some oxidative stress-related terms (i.e., reactive oxygen species metabolic process GO:0072593, hydrogen peroxide metabolic process GO:0042743) (Extended Data Fig. 1). Figure 1d presents expression data for the 31 significant (FDR adjusted p-value < 0.1) annotated DEGs associated with the immune response GO term. This group included Nv homologs of Notch and Elk1, and members of the complement system. Samples clustered according to feeding regime, with the exception of the fed clone 3 and starved clone 4 (anemones 3F and 4S).
Starvation increases the susceptibility of N. vectensis polyps to bacterial infection
Given that GO terms associated with immunity were underrepresented in starved anemones, we hypothesized that Nv’s ability to withstand bacterial challenge would be reduced in starved anemones. Pseudomonas aeruginosa is a Gram-negative bacterium that is pathogenic in a variety of hosts including some plants, invertebrates, and vertebrates31,32. Recently, P. aeruginosa strain PA14 was also shown to be pathogenic for juvenile Nv25. To determine if fed and starved anemones have different susceptibility to P. aeruginosa, we infected a total of 60 fed and 60 starved 40-day old juvenile anemones over three separate experiments with an average of ∼4.5 × 108 CFU/ml of PA14. We then visually monitored disease progression daily based on tissue degradation and death. We found that starved anemones died at a significantly faster rate than their fed counterparts (Kaplan-Meier p > 0.01, N = 24, Fig. 2), and that this pattern was reproducible across trials (Extended Data Fig. 2 a & b for two additional trials). Therefore, consistent with reduced expression of immunity-related genes in adults, starved juvenile Nv have increased susceptibility to the effects of a bacterial pathogen.
NF-κB protein and DNA-binding activity are reduced in starved anemones
Transcription factor NF-κB has a broad role in immunity across Metazoa21,33,34. We have also previously shown that NF-κB protein is expressed in juvenile and adult Nv, with expression starting as early as 30 h post-fertilization22,35. Given the upregulation of NF-κB following infection with P. aeruginosa25 and that several immunity-related genes were downregulated in our starved anemone gene expression data, we aimed to determine whether starvation had an effect on NF-κB signaling. We first hypothesized that NF-κB transcripts would be downregulated under starvation in Nv, and while gene expression patterns in fed and starved adult Nv showed that NF-κB transcripts were downregulated in starved anemones (log2Fold Change = -0.96, raw p-value = 0.01), this trend was not significant after FDR correction.
We next compared NF-κB protein and DNA-binding levels in starved vs fed adult anemones. To do this, we again generated clonal pairs (N = 3) of animals (by bisection and regeneration), which were then fed or starved for two weeks. To compare NF-κB protein levels, Western blotting was performed on whole animal extracts from fed and starved clonal pairs. On average, starved anemones had ∼70% less NF-κB protein than their fed counterparts (Fig. 3a). To compare NF-κB DNA-binding activity, an electromobility shift assay (EMSA) was performed using extracts from three fed vs starved anemone pairs and a 32P-labeled κB-site probe that we have previously shown can be specifically bound by Nv-NF-κB36. Consistent with the decrease in protein levels, starved anemones had less NF-κB DNA-binding activity than their fed counterparts (Fig. 3b).
Because of the increased susceptibility of starved juvenile anemones to bacterial infection (Fig. 2), we were interested in determining whether the overall decrease in NF-κB protein during starvation was also observed in juvenile Nv. To do this, we performed anti-Nv-NF-κB Western blotting and immunohistochemistry on 40-day old Nv that had been fed regularly or never fed. To analyze Nv-NF-κB protein levels in juvenile Nv, we pooled 100 fed or starved polyps, lysed each pool by boiling in SDS sample buffer, and analyzed those lysates by Western blotting. Results showed that starved polyps had ∼20% less NF-κB protein than fed anemones (Fig. 3c). In addition, starved juveniles had ∼60% fewer detectable NF-κB-positive cells by immunohistochemistry (Unpaired t-test p < 0.05) (Fig. 3d; Extended Data Table 2). Taken together, these results indicate that starvation causes the downregulation of Nv-NF-κB in adult anemones, which results in decreased NF-κB protein and DNA-binding activity, and that decreased NF-κB protein expression is also seen in juvenile anemones, which have increased susceptibility to bacterial infection.
Gene co-expression network analysis reveals possible cnidarian immune gene network
NF-κB is known for its role as master regulator of immunity33,34; this role can include the regulation of other members in the NF-κB signaling pathway. One such member, TNF receptor-associated factor 3 (TRAF3), has been previously observed to be upregulated with NF-κB in Nv25. Indeed, we found the Nv TRAF3 gene has three predicted strong NF-κB sites within 1000 bp of its transcription start site (TSS) (Extended Data Fig. S5). All three of these predicted NF-κB sites can be bound by bacterially expressed Nv-NF-κB in an electrophoretic mobility shift assay (Fig. 4a). Furthermore, a luciferase reporter plasmid containing the upstream promoter region of Nv-TRAF3 can be activated by Nv-NF-κB when co-transfected in HEK 293 cells (Fig. 4b), and mutation of the three NF-κB binding site abolished its ability to be activated by Nv-NF-κB (Fig. 4b).
To gain a broader overview of a possible Nv-NF-κB gene expression network that is relevant to the starvation response, we used a systems biology-based approach wherein we identified modules of genes whose expression profiles were correlated with NF-κB expression. For this analysis, we used the R package WGCNA (Weighted Gene Correlation Network Analysis). This approach generated module eigengenes, or representative gene expression profiles for each module, and we identified the module containing NF-κB (“Green” module). This “Green” module, which contained 1317 genes, had a correlation coefficient of -0.66 with starvation (Extended Data Fig. S3), indicating that expression of genes in this module tended to be downregulated by starvation. We next ranked these transcripts by ‘membership score’ (kME), which is a measure of how strongly each gene corelates with the module’s eigengene. Figure 4c shows a heatmap of the 50 genes in the “Green” module with the highest membership scores. These 50 genes (36/50 annotated) included ones encoding a TRAF3 homolog and autophagy-related protein 2 homolog A, among others.
To further characterize the genes in the “Green” module, we performed a binary analysis of GO enrichment and found enrichment for terms related to Biological Processes including immunity (i.e., immune response, regulation of immune response GO:0050776, antigen processing and presentation), cell signaling (i.e., regulation of IKK/NF-κB signaling GO:0043122, JNK cascade GO:0007254, MAPK cascade GO:0000165), and cell death (i.e., cell death GO:0008219, negative regulation of cell death GO:0060548, regulation of necrotic cell death GO:0010939) (Extended Data Fig. 4). Additionally, we found that 25 of these top 50 genes had predicted NF-κB binding sites within 1,200 bp of their TSS (Fig. 4c, Extended Data Table S3). Of note, these predicted Nv-NF-κB binding sites have high affinity DNA binding, based on their high Z-scores (Extended Data Table S3) from our previous analysis of DNA binding by Nv-NF-κB using protein-binding microarrays27.
Discussion
Here, we demonstrate a correlation between decreased nutritional status and decreased immunity in the sea anemone Nematostella vectensis (Nv). We have found that feeding status has a significant impact on gene expression, in addition to the effect of genetic background, consistent with what is seen in other cnidarian gene expression studies involving clonal populations37,38. Furthermore, we have shown that the levels and activity of immunity-related transcription factor NF-κB are also reduced under starvation conditions. Thus, we demonstrate a link between nutritional status and immunity in a cnidarian, suggesting that a nutrition-immunity axis has a long evolutionary history. These results also have implications for other cnidarians, e.g., corals, which are endangered by rapidly changing environmental conditions.
Gene expression data and GO enrichment analysis provide insight into the transcriptional effects of starvation in our cnidarian model. Downregulation of terms such as response to nutrient, lipid metabolic process, carbohydrate metabolic process, glycosylation GO:0070085, and proteolysis GO:0006508 under starvation conditions suggest an exhaustion of energetic sources. Downregulation of genes associated with metabolism of different energy sources (carbohydrates, lipids, proteins) during periods of starvation has been previously observed in other species4,39-42. These downregulated GO terms indicate that the anemones in our experiments were indeed starved. Moreover, downregulated terms including ATP biosynthetic process GO:0006754, proton transmembrane transport, and electron transport chain GO:0022900 also support the energetic shortage experienced by Nv under starvation. Generally, animals that encounter prolonged periods of food deprivation exhibit low metabolic rates1, and so it is perhaps not surprising that our data showed downregulation of metabolic processes under food limitation.
We also noted a significant down-regulation of genes and GO terms associated with immunity during starvation of adult Nv. Interestingly, food limitation has been previously observed to reduce immunity against bacteria in the caterpillar Manduca sexta, as well as decreasing its resistance to oxidative stress3, which is a pattern consistent with our GO enrichment for starved Nv. Overall, starved Nv DEGs showed underrepresentation of GO terms associated with immunity (i.e., immune response, defense response, and antigen processing and presentation), suggesting a diminished pathogen defense and underrepresented GO terms associated with response to stress (i.e., reactive oxygen species metabolic process, hydrogen peroxide metabolic process), suggesting a vulnerability to oxidative stress.
Starved Nv polyps had a reduced ability to withstand infection by P. aeruginosa, which was correlated with an overall decrease in NF-κB protein levels, as judged by both Western blotting and immunostaining. This relationship between starvation and susceptibility to pathogen infection has been observed in invertebrates3-5,11, mice7,9, and humans8,10,43. A similar relationship has also been observed in Apis mellifera ligustica (Italian honeybee), wherein dietary supplementation with an essential fatty acid improved their ability to withstand bacterial infection and resulted in transcriptional upregulation of the NF-κB pathway genes Toll, Myd88, and Dorsal (NF-κB homolog)11. The underlying concept shared by all of these examples, as well as our results with Nv herein, is that the immune response is an energetically demanding process, which has led to the evolution of proper resource allocation under different nutritional states. For example, Drosophila diverts energy from growth and nutrient storage when Toll signaling is activated44, and parasitic infection in Bombus terrestris (Bumblebee) becomes more virulent under low-nutrient conditions45.
Many tropical reef building corals derive the majority of their energy from intracellular symbiotic algae46; however, this symbiotic relationship can be lost under a variety of stressors in a process known as coral bleaching47,48. Evidence suggests that feeding via heterotrophy is important for corals to mitigate bleaching in the face of warming oceans17,18. For example, the branching coral Montipora capitata shows enhanced recovery from bleaching compared to other corals by increasing the amount of carbon it acquires through heterotrophy19. Similarly, the coral Pocillopora meandrina incorporates more heterotrophic carbon when there is more food locally available49. Combined with the results presented herein, cnidarians appear to have complex and dynamic ways to respond to stress in the midst of poor nutrient availability.
In addition to bleaching, coral infectious diseases appear to be increasing, which could be due to environmental effects on immunity. Over the last 50 years, ∼40 different coral diseases have been described50, with the latest source of concern being Stony Coral Tissue Loss Disease (STCLD) that is affecting Caribbean corals51,52. Previous work by our group showed that symbiosis with Symbiodiniaceae algae in the anemone Exaptasia pallida has a negative correlation with anemone NF-κB levels, suggesting that the symbiotic state decreases its NF-κB-dependent immunity27. Thus, the survival of some cnidarians under certain environmental stressors may be linked to nutrition and immunity.
The phylum Cnidaria emerged approximately 700 million years ago53. Since then, individual cnidarians have likely evolved unique genes as part of their immune systems. By taking a systems biology approach, we were able to identify modules of genes that were highly correlated with starvation status in Nv. We also identified a module of genes to which NF-κB belongs that was composed of 1317 genes, of which ∼30% are unannotated. Given that many genes in this module are associated with immunity, cell signaling, and cell death, it is likely that some unannotated genes within this same module play roles in immunity, such as being direct anti-microbial effectors. Moreover, we found that 25 of the top 50 genes in the same module as NF-κB had one or more predicted NF-κB-binding sites identified using a PBM-based Nv-NF-κB motif27. These results provide avenues to explore novel basal immune gene interactions and are consistent with an evolutionarily conserved role of NF-κB in immunity-related gene regulation.
Previous work in Nv identified an unannotated anti-microbial gene that is upregulated in response to the immune stimulatory molecule 2’3’-cGAMP25, however, that gene was not affected by starvation-induced downregulation of NF-κB, suggesting that it is not a direct NF-κB target gene. In contrast, several lines of evidence suggest that TRAF3 is a direct target of Nv-NF-κB: 1) there are three strong NF-κB binding sites located within 1000 bp of the TRAF3 TSS (Extended Data Fig. 4; Fig. 4a), 2) Nv-NF-κB can activate a reporter locus containing the upstream promoter region of TRAF3 and this activation requires the upstream NF-κB binding sites (Fig. 4b), and 3) Nv-TRAF3 is upregulated by activation of the c-GAS-STING pathway in Nematostella25. It is of interest that mammalian TRAF3 is a regulator of NF-κB and has a broad role in B-cell immune activation and survival54. A correlation between NF-κB and TRAF3 has been reported in several other cnidarian studies. First, NF-κB and TRAF3 are coordinately induced to high levels in the stony elkhorn coral Acropora palmata following acute heat stress55; and in Nv following 2’3’-cGAMP stimulation of the c-Gas-STING pathway25. Additionally, TRAF3 has been suggested to play a role in coral heat stress response56,57, and has been proposed to be a NF-κB target gene in heat-stressed E. pallida58. Therefore, our results provide a dataset to explore new gene network interactions, as well as leading to the identification of unannotated gene transcripts that are involved in the cnidarian immune system, some of which may be previously unknown anti-microbial agents.
Overall, we show a link between nutrition and immunity in Nv, and that NF-κB may play a role in this relationship. These data provide a model for better understanding the interplay between nutrition and certain diseases in cnidarians. The continued study of these important pathways in basal metazoans will further our understanding of where and how these pathways originated, as well as implications for the physiological effects in critical marine organisms as we move into an era of changing climate.
Materials and Methods
Care, husbandry and cloning of Nematostella vectensis
N. vectensis from a Maryland population were obtained from Mark Martindale and Matt Gibson, and spawnings were performed as previously described12,23,59,60. Adults, polyps, and larvae were maintained in 1/3 strength artificial sea water (1/3 ASW: ∼12 parts per 1,000) in a dark incubator at 19°C. Adult anemones were fed freshly-hatched brine shrimp (Artemia) and young polyps were fed ground Artemia in 1/3 ASW three times per week. Water changes were performed weekly for all anemones. To generate clonal pairs, adult animals were allowed to fully relax and were then bisected perpendicularly. Halves were placed into separate wells, and anemones were allowed to regenerate for 30 days. Feeding was paused for all animals during the 30-day regeneration period, i.e., to allow the tentacles to form from the aboral end. Thereafter, both members of the clonal pair were fed in equal amounts.
RNA extraction and preparation for TagSeq on fed vs. starved anemones
Clonal pairs of adult anemones were generated by bisection and the halves were allowed to heal for 30 days as described above. Thirty days was chosen to allow injury and regeneration-related genes to return to basal levels, as demonstrated previously13,14. Clonal pairs were fed equal amounts of food on a regular schedule during healing once all previously aboral ends had developed tentacles. Clonal pairs were split to be fed or starved for 30 days before being flash-frozen on dry ice prior to RNA extraction. Total RNA was isolated from eight clonal pairs with RNAqueous™ Total RNA Isolation Kit (Invitrogen) according to the manufacturer’s instructions, with additional grinding using a plastic pestle during tissue lysis. Next, DNA was eliminated using DNA-free™ DNA Removal Kit (Invitrogen). RNA quality was assessed by agarose gel electrophoresis, checking for the presence of ribosomal RNA bands. RNA concentrations were quantified using a NanoDrop ND-1000 Spectrophotometer. Samples were then normalized to 728 ng of total RNA for submission to the University of Texas at Austin – GSAF’s TagSeq Service. Libraries were created by the GSAF and sequenced on a NovaSeq 6000 SR100.
Transcriptome read mapping
Reads were processed following the TagSeq pipeline (https://github.com/z0on/tag-based_RNAseq). In brief, adapters and poly(A)+ tails were trimmed using Fastx_toolkit and sequences <20 bp with <90% of bases having quality cut-off scores >20 were trimmed. PCR duplicates sharing degenerate headers were also removed. Resulting quality-controlled reads were aligned to the Nematostella transcriptome60 using Bowtie2.2.062.
Differential gene expression and gene ontology analyses
Differential gene expression analysis was performed using DESeq2 v.1.30.163 in R v.4.0.464. The arrayQualityMetrics65 package tested for outliers, which were defined as any sample failing two or more outlier tests; no outliers were identified. Significant DEGs were identified as those with an FDR-adjusted p-value < 0.1. Expression data were normalized using the rlog function within the package vegan66, and normalized data were then used for principal component analysis (PCA) to characterize differences in gene expression between starved and fed (control) groups. Significance was tested by PERMANOVA using the adonis function as part of the vegan package66.
Gene Ontology (GO) enrichment analysis was performed using Mann-Whitney U tests (GO-MWU) based on ranked p-values67. GO enrichment results based on the ‘molecular function’ overarching division were plotted as dendrograms with GO categories clustering based on shared genes. Fonts and colors were used to indicate significance and direction of change respectively. To generate a heatmap of “Immune Response”-annotated genes, we used the package pheatmap68 to showcase differences in expression relative to mean expression across samples.
Bacterial challenge of N. vectensis
Approximately two-week-old polyps were placed into single wells of a 24-well plate, and they were then either fed ground-up Artemia for 30 days or starved until infection was initiated. Infection was performed essentially as described previously25. Single colonies of P. aeruginosa strain PA14 were cultured overnight in Luria Broth, bacteria were centrifuged for 10 min at 1627 x g, rinsed once with 1/3 ASW, centrifuged again, combined, and resuspended to an OD600 of ∼0.1 in 1/3 ASW. A small aliquot was taken for plating to calculate CFU/ml. Polyps were infected by placing them in the well of a 24-well plate containing PA14 (1 ml). Survival was monitored daily, mortality was determined based on tissue degradation and the absence of response to light and touch cues24. Infection was performed three separate times; once with 12 anemones per feeding regime, and twice with 24 anemones per treatment regime.
Tissue lysis of N. vectensis
Whole protein lysates from anemones were generated following a protocol described previously69. Briefly, adult anemones (about 2-cm long) were homogenized using a plastic pestle in 1.5-ml microcentrifuge tubes containing 150 μl of ice-cold AT Lysis buffer with proteinase inhibitors (HEPES [20 mM, pH 7.9], EDTA [1 mM], EGTA [1 mM], glycerol [20% w/v], Triton X-100 [1% w/v], NaF [20 mM], Na4P2O7·10H2O [1 mM], dithiothreitol [1 mM], phenylmethylsulfonyl fluoride [1 mM], leupeptin [1 μg/ml], pepstatin A [1 μg/ml], aprotinin [10 μg/ml]). Cell lysis was enhanced by sonicating 5 times for 10 sec on setting 3 with 1 min on ice in between, samples were then passed five times through a 30-gauge needle. NaCl was added to a final concentration of 150 mM. Finally, samples were centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant was stored at -80°C until needed. For protein lysates from juvenile polyps, 100 40-day old polyps were pooled into a centrifuge tube. Sea water was removed through aspiration and 50 μl 4x SDS sample buffer (Tris-HCl [0.25 M, pH 6.8], SDS [2.3% w/v], glycerol [10% w/v], β-mercaptoethanol [5% v/v], bromophenol blue [0.1% w/v]) was added to the tube. Samples were heated at 95°C for 10 min with vortexing halfway through and at the end. Finally, 50 μl distilled H2O was added to the samples.
Western blotting and electrophoretic mobility shift assay (EMSA)
Western blotting for Nv-NF-κB was performed as previously described22,26,27,69. Briefly, proteins were separated on a 7.5% SDS-polyacrylamide gel. Proteins were then transfered at 4°C to a nitrocelluolose membrane at 250 mA for 4 h and then 170 mA overnight. Nitrocellulose membranes were incubated in blocking buffer TBST (Tris-HCl [10 mM, pH 7.4], NaCl [150 mM], Tween 20 [0.1% v/v]) with powdered milk (5% w/v) (Carnation) at room temperature for 1 h. Membranes were then incubated in anti-Nv-NF-κB antibody22 (diluted 1:10,000 in blocking buffer) overnight at 4°C. Membranes were washed several times with TBST before incubating with a horseradish peroxidase-conjugated anti-rabbit secondary antiserum (1:4,000, Cell Signaling) for 1 h at room temperature. Membranes were then treated with SuperSignal West Dura Extended Duration Substrate (Pierce), and blots were imaged on a Sapphire Biomolecular Imager. The same filters were also stained with Ponceau S stain to ensure approximately equal total protein loading.
EMSA was performed as previously described22,26,27,69 using a 32P-labeled κB-site DNA probe (GGGAATTCCC) and adult anemone tissue lysates (described above). Lysates and the κB-site probe were incubated in binding buffer (HEPES [10 mM, pH 7.8], KCl [50 mM], DTT [1 mM], EDTA [1 mM], glycerol [4% w/v]) with poly dI/dC (40 ng) and BSA (200 ng) at 30°C for 30 min. Supershifts were performed by incubating samples with 2 μl of anti-Nv-NF-κB antiserum, after binding to the DNA probe, for 1 h on ice. EMSA gels were dried and then imaged on a Sapphire Biomolecular Imager.
The EMSA for TRAF3 promoter region was performed as above, except using bacterially expressed GST-Nv-NF-κB, which was purified as described previously27. Purified GST-Nv-NF-κB was then incubated in binding buffer as described above with each of the following 32P-labeled probes:
(1) 5’-TCGAGAGGTCGGGAAAGCCCCCCCCCG-3’
(2) 5’-TCGAGAGGTCGGGAAACCCCCCCCCCG-3’
(3) 5’-TCGAGAGGTCGGGGAACTCCCCCCCCG-3’
Underlined sequences are predicted NF-κB binding sites in the Nv TRAF3 promoter (Extended Data Fig. 5). The dried EMSA gel was exposed overnight to X-ray film at -80°C overnight. Film was developed using a standard X-ray film developer.
Immunohistochemistry of N. vectensis polyps
Immunohistochemistry was performed as previously described22,35,69. Polyps were fixed in formaldehyde (4%) in 1/3 ASW overnight at 4°C and washed three times with PTx (Triton X-100 [0.2% v/v] in PBS). Antigen retrieval was done by microwaving samples in warm urea (5% w/v) at the lowest setting for 5 min. Samples were cooled at room temperature for 20 min. Samples were then washed three times with PTx. Samples were moved to blocking buffer (PTx + normal goat serum [5% v/v] + BSA [1% w/v]) and allowed to permeabilize overnight at 4°C on a nutator. Blocking buffer was replaced with anti-Nv-NF-κB primary antiserum diluted in blocking buffer (1:100) and incubated overnight at 4°C. Samples were then washed four times with PTx and incubated in Texas-red-conjugated anti-rabbit secondary antiserum (1:160, Invitrogen). Polyps were then washed four times with PTx. Nuclei were stained by adding DAPI to a final concentration of 5 mg/ml. Samples were imaged on a Nikon C2+ Si confocal microscope. NF-κB-positive cells were counted using the Cell Counter plug-in in ImageJ.
Luciferase reporter gene assay
Luciferase reporter gene assays were performed in HEK 293 cells as previously described22. Cells were plated in 6-well 35-mm plates to 60% confluence and transfected with: 0.5 μg of (i) pGL3-Nv-TRAF3, which consists of pGL3 with 1,220 bp of the promoter region of Nv-TRAF3 cloned upstream of the luciferase gene; or (ii) pGL3-Nv-TRAF3-3X-mut, similar to (i) but with all three putative κB-binding sites mutated to 5’-”GGGGAAAGCTT”-3’, and 2 μg of (i) a Nv-NF-κB expression plasmid or (ii) a pcDNA empty vector. Every transfection was performed with 15 μg of PEI. Two days after the transfection, cells were lysed with Reporter Lysis Buffer (Promega) following manufacturer’s instructions.
Weighted correlation network analysis (WGCNA)
Weighted Gene Correlation Network Analysis was performed using WGCNA70 and genes with low basemean values (<3) were removed and all remaining data were rlog-normalized. Outlier samples were checked within the WGCNA package, and no outliers were detected. Unsigned connectivity between genes was determined and eigengene expression of these modules were correlated to feeding conditions. The “Green” module was chosen by manually searching modules for the NF-κB transcript. Gene Ontology (GO) enrichment analysis was performed as described in the ‘Differential Gene Expression Analysis’ text with the modification of using continuous kME values instead of -log p-values. To generate module heatmap, genes with the highest module membership scores (kME values) within specific modules (e.g., “Green” module) were identified and relative expression was plotted using the package pheatmap68.
To identify genes with putative NF-κB binding sites, the transcripts of the top 50 “Green genes” (Fig. 4c) were aligned to the N. vectensis genome71,72 on Ensembl73 using BLAST to identify genomic location. We then extracted 1,200 bp upstream of the TSS of every matched gene. To identify Nv-NF-κB binding sites in these upstream regions, we used the program FIMO74 (with a p-value cutoff of 1E-04) and a DNA site motif based on Nv-NF-κB DNA binding from a Protein Binding Microarray (PBM) 27.
Competing interest statement
The authors declare no competing interest.
Author contributions
P.J.A.C. performed immunohistochemistry, infection experiments, Western blotting and EMSA of Nv lysates. P.J.A.C. and N.D. performed Western blots and EMSAs. P.J.A.C., J.F., and S.W.D. contributed to writing scripts used in this manuscript. J.J.B. performed luciferase assays and TRAF3 EMSA. P.J.A.C., S.W.D., and T.D.G. designed experiments and wrote the manuscript.
Acknowledgments
This research was supported by National Science Foundation grants IOS-1354935 and IOS-1937650 (to T.D.G. and S.W.D.). P.J.A.C. was supported in part by a Warren McLeod Marine Fellowship, and N.D. was supported by the Boston University Undergraduate Research Opportunities Program. We thank Mark Martindale (Whitney Laboratory for Marine Science) and Matt Gibson (Stowers Institute for Medical Research) for sharing Nv with us, Stephen Lory (Harvard University) for the PA14 strain, and Trevor Siggers (Boston University) for help with the analysis of Nv-NF-κB DNA-binding sites.