This time series RNA-seq experiment was conducted to investigate the molecular innate immune response of the symbiotic Exaiptasia pallida strain CC7 to Vibrio parahaemolyticus strain O3:K6 infection over 12 hours. Analyses focus on three main aspects of the innate immune response of the host: 1) the genes differentially expressed in infected anemones, 2) the gene expression changes over the onset of infection, and 3) the comparison between the responses to Vp and exposure to the endotoxins LPS. Punctual pairwise gene expression and time series analyses of Exaiptasia’s transcriptomic response to the virulent pathogen Vp identified 6,842 differentially expressed contigs responsive to bacterial infection across four time points. Below we discuss pathway level kinetics that contribute pertinent information to previously described innate immune gene families as well as novel innate immune gene candidates.
Physiological response to bacterial challenge
Ep exposed to the clinical O3:K6 Vp strain at 108 cfu/ml starts to show mortality after 24 hours. Similar to this cross-pathogenicity, another study using the gram-negative human pathogen - Serratia marcescens - at the same concentration showed that Ep has a survival rate of 70% after 24 hours (Krediet et al. 2014). Our observations of Ep’s physiological response to Vp also concur with the descriptions of the same species infected with the coral pathogens Vc and V. shiloi (Zaragoza et al. 2014). Although darkening of the tissues during those infections was linked to melanin production, our search for evidence of melanin biosynthesis at the transcript level failed to identify homologs in this dataset. Anemones exposed to Vp show darkening of their tissues, retraction of tentacles, and mortality after 24 hours post-inoculation. Thus, our results suggest that the clinical Vp strain O3:K6 responsible for gastroenteritis in humans remains pathogenic in saltwater at 27 degrees Celsius, and can successfully infect and start killing aiptasia anemones after 24 hours. Brown and Rodriguez-Lanetty (2015) determined that an exposure to 108 cfu/ml of Vc at 30 °C for three days was a sub-lethal treatment from which 100% of Ep exposed could recover. A similar result was observed here but after only 12 hours of exposure to 108 cfu/ml of Vp at 27 °C. Based on these results, Ep likely possesses an innate immune system capable of efficiently fighting back seriously challenging and varied pathogenic assaults.
Functional support for non-canonical cytoplasmic PRRs
Within cells, intracellular PRRs detect pathogens and are critical in the response to microbial components in the cytosol. Here, homologs of NLRs, NLRC-like genes were significantly regulated at each time point (Supplementary Table 4) and include up-regulated sequences with NACHT-like domains and LRR as well as a down-regulated sequence with trans-membrane domains (TMD). The NLRs are cytoplasmic proteins, which recognize bacterial peptidoglycans and trigger proinflammatory and antimicrobial immune response. Among the NLR family, the NLR family CARD domain containing protein 4 (NLRC4) is the best-known mammalian member capable of triggering the inflammasome upon detection of microbial ligands (Duncan and Canna 2018). Interestingly, a large and complex NLR repertoire is present in early-diverging metazoans, including cnidarians, which exhibit an especially rich family of NACHT/NB-ARC containing genes in comparison with many later-diverging metazoans (Lange et al. 2011; Hamada et al. 2013). In actinarians, the diverse repertoire of NLR-like genes and their novel domain architecture has been hypothesized to perform novel roles in immune signaling and/or pathogen recognition, which are yet to be described (Baumgarten et al. 2015; van der Burg et al. 2016). Among the 78 NLRC contigs regulated during Ep’s infection, some contain LRR motifs (Supplementary Table 4), suggesting a capacity to bind to PAMP. These genes warrant further functional investigations to determine whether they play a role in the recognition of pathogens within the cells.
Additional genes were identified as potential cytoplasmic PRRs, including interferon-induced helicase C domain-containing protein 1 (IFIH1) and RIG-I-like receptor 3 (RLR-3), which both contain the RIG domain of the RLRs and play a role in the intracellular recognition of viruses. In total eight IFIH1 sequences and three RLR-3 sequences were detected as up-regulated at 3, 6 and 12 hours (Supplementary Table 4). As the pathogen Vp acquired several new genomic islands through horizontal transfer, which contain phage-encoded virulence factors (Ceccarelli et al. 2013), it is possible that the Ep genes mentioned above are induced by viral-like genetic material brought into the host cells by invading Vp. To our knowledge, this is the first report of infection-induced RLR in a cnidarian.
Involvement of the lectin-complement pathway
In the response to Vp infection, we found that anemones up-regulated Ficolin-1 homologs containing TMD and Ig (unlike bilaterian ficolins; van der Burg et al. 2016) at 3, 6, and 12 hours (Supplementary Table 4), as well as several components of the lectin-complement pathway. For example, although no regulation of MASP was detected, complement component C2, C3 and C4 were up-regulated over time. Moreover, aiptasia’s factor B (Ep_Bf-1) of the alternative pathway was up-regulated in the infected anemones at 3, 6 and 12 hours (Supplementary Table 4). Poole et al. (2016) used quantitative real-time PCR to show up-regulation of both Ep’s Bf-1 and MASP genes in response to 24-hour exposure to low and high concentrations of the pathogen Serratia marcescens, and concluded that concentration of microbes, and symbiotic state influence complement gene expression. The co-expression of TMD/Ig-containing Ficolin-1 and genes of the lectin-complement pathways detected here supports a potential role of CniFLs in the initial recognition of pathogens (Baumgarten et al. 2015), and highlights both the lectin and alternative pathways active roles in the defense of anthozoans against pathogenic bacteria. Nevertheless, it remains to be demonstrated whether the CniFLs bind to certain PAMP and can trigger the lectin-complement pathway, potentially stimulating phagocytes in aiptasia.
Several other lectins were detected during the response to Vp infection. For example, L-rhamnose-binding lectins (Ep_RBLs) contigs, homologs of collectin-12 (colec12), and a single C-type lectin LRR-containing sequence were up-regulated (Supplementary Table 4). RBL interact with various types of bacteria in fish and invertebrates to induce proinflammatory cytokines such as IL-1β, TNFα and IL-8, and enhance macrophage phagocytosis (Watanabe et al. 2009). The scavenger receptor lectin-like family is rich and diverse in anthozoans (Neubauer et al. 2016), plays an important role in host-symbiont interactions during onset of symbiosis (Davy et al. 2012), and can bind to pathogens (Kvennefors et al. 2008). Our results highlight the diversity of lectins potentially involved in the recognition, potential activation of phagocytosis, and proinflammatory signaling upon contact with a pathogen in aiptasia.
TLR-like pathway without TLR
Here, neither homologs of canonical vertebrate TLR or IL-1R, nor orthologs of the Hydra’s HyTRR and HyLRR genes (Augustin et al. 2010) were identified in the Ep transcriptomic responses. TLR and IL-1R-like homologs have been identified in several other anthozoans (Poole and Weis 2014), however many of those genes lack the ectodomain LRR and only possess TMD- and TIR-domains (Baumgarten et al. 2015). Aiptasia’s genome possesses homologs of the major PRR types including NLRs, RLRs, and C-type lectin receptors (CLRs) but lack TLRs. However, TLR-to-NF-κB pathway components are definitely present in Ep, as well as in A. digitifera and O. faveolata corals, Hydra and Nematostella (reviewed in Brennan and Gilmore 2018). Several of these genes include homologs with well conserved domains such as MyD88, TRAF6, IRFs, AP-1, and NF-κB, and were positively responsive to Vp infection (Supplementary Table 4). Functional conservation of the TLR-to-NF-κB was confirmed in Nematostella (Brennan et al. 2017) and O. faveolata (Williams et al. 2018). Furthermore, in Hydra, MyD88-deficient polyps were more susceptible to infection with the human pathogen Pseudomonas aeruginosa (Franzenburg et al. 2012). Taken together, these results suggest that in Exaiptasia, the recruitment of MyD88 and downstream signaling to activation of NF-κB is induced by Vp, and like in Hydra must involve different PRRs than prototypical TLR homologs. Based on these findings, we hypothesize that an ancestral TLR-to-NF-κB signaling pathway responsive to bacterial stimuli evolved before transmembrane TLRs in early metazoans.
Potential TNFR signaling to inflammation and apoptosis
Another important aspect revealed by this study is the activation of cytokine-dependent signaling pathways in the innate immune response to Vp, two of which could lead to apoptosis and an inflammatory response. Indeed, the regulation of the ‘TNF-mediated signaling pathway’ was enriched at 3, 6 and 12 h post infection. One of these pathways likely starts with the activation of the TNFR1, which is the receptor for the proinflammatory cytokine TNFα and the cytotoxic protein lymphotoxin-alpha in humans. The Ep_TNFR1 DEC (GenBank: KXJ17455) is up-regulated 223.9 times after 1 hour, then again at a lower level after 3, 6 and 12 hours. Several TRAFs including TRAF2, -5 and the MAP3K14 among other MAPK3 homologs, are potentially acting downstream of Ep_TNFR1 and are up-regulated at 6 and 12 hours. Notably, more support for the TNFR1 pathway is shown with the TNFAIP3 and the TNIP1 homologs that are both up-regulated at 3, 6, 12 hours. TNFAIP3 is an essential component of the ubiquitin-editing protein complex that ensures the transient nature of inflammatory signaling pathways, while TNIP1 inhibits the NF-κB activation and TNF-induced NF-κB-dependent gene expression by regulating the activity of TNFAIP3. Finally, the transcription factor AP-1 is up-regulated at each time point and may influence the immune response via activation of Ep_TNFR1.
The other cytokine-dependent pathway may involve the TNFRSF-mb16 (GenBank: KXJ29589) homologs, which contain a Death domain and are up-regulated at 3, 6 and 12 hours. The Ep_TNFRSF-mb16 sequence resembles the Fas receptor (FasR or TNFRSF6 gene) which plays a central role upon Fas ligand (FasL) binding in the physiological regulation of programmed cell death in humans via its interaction with FADD and CASP8. In the coral A. digitifera, thirteen TNFR-like sequences containing the DD were identified and proposed as mediators of apoptosis through caspase activation (Quistad and Traylor-Knowles 2016). Moreover, the conservation of the TNF-induced apoptotic response was demonstrated in the coral Acropora digitifera (Quistad et al. 2014). We also identified candidates potentially acting up- and down-stream of FasR, such as matrix metalloproteinase and FasLG homologs.
Apoptosis as defense mechanism
In aiptasia's response to infection, a functional enrichment for ‘regulation of apoptotic process’ could be detected at 3 hours. The major cell death pathways, including apoptotic cell death, are a crucial barrier against microbial infection (Bergsbaken et al. 2009; Lamkanfi and Dixit 2010; Zitvogel et al. 2010). For instance, in response to bacterial infection, apoptosis or programmed cell death is used in the host innate immune response to: 1) eliminate pathogens at the early stage of infection without emitting alarm signals, and 2) induce dendritic cells (DCs) to engulf apoptotic bodies containing infected microbes (Elliott and Ravichandran 2010). Among the 216 DECs supporting the enrichment, many key players of apoptosis as well as genes with potential connections to the apoptotic pathway were identified at each time point. For example, the Ep_CASP8 is up-regulated at 3 and 6 hours and likely functions within the cells. If interacting with the Ep_TNFRSF proteins, Ep_CASP8 would represent the extrinsic (receptor-mediated) pathway to apoptosis. On the other hand, the intrinsic (mitochondria-mediated) pathway is strongly supported by the up-regulation of ATF4, Bcl-2, Bcl-W, Bax, Bak, Apaf-1 as well as CASP9 homologs in Ep (Supplementary Table 4). Both pathways could engage the apoptotic executor - CASP3, which is up-regulated for the duration of the infection with a peak of 1,250 in fold change at 6 hours. These results strongly suggest that apoptosis plays an important role in the innate immune response of Ep against bacterial pathogens. As Ep is capable of recovering from a punctual exposure to Vp, it is probable that apoptosis plays a part in the removal of invading bacteria and the survival of the host. Further work will determine if and how Ep is capable of containing a bacterial invasion via programmed cell death.
Comparison between responses to Vp infection and LPS
The genes of the TLR-to-NF-κB like pathway identified in the Ep’s response to Vp infection were not activated by LPS. Hydra polyps exposed to LPS treatments activate the expression of the antimicrobial peptides in a dose-dependent manner, suggesting that LPS is an inducer of immune defenses in cnidarians (Bosch et al. 2009). Indeed, studies on corals have shown that LPS can trigger an immune response in several species (Fuess et al. 2016, 2017; Kvennefors et al. 2010), and more specifically genes of the TLR-to-NF-κB pathway (Williams et al. 2018; Connelly et al. 2020). However, LPS failed to stimulate an engineered Hydra LRR protein to activate NF-κB signaling in human cells, whereas flagellin did (Bosch et al. 2009). Similarly, LPS did not stimulate the sole Nematostella TLR to activate a reconstituted Nv-TLR-to-NF-κB pathway in 293T cells, while flagellin and heat-inactivated Vc did (Brennan et al. 2017). Finally, in an attempt to induce innate immune response in Exaiptasia, Poole and colleagues (2016) exposed anemones to LPS and peptidoglycan, but did not detect any significant changes in gene expression, and decided to use live Serratia marcescens instead. Interestingly, in this study the LPS-induced TNFα factor was up-regulated in response to both LPS and Vp. Overall, these results suggest that in cnidarians lacking TLR homologs, LPS can still trigger the production of cytokines and antimicrobial peptides independently of the TLR-to-NF-κB pathway.
Novel genes responsive to infection
Several of the genes similar to the potential novel actinarian immune genes (NG1, NG2, and NG3) proposed by van der Burg et al. (2016) based on domain architectures, are here annotated F-box only protein 11, 4 and F-box/LRR-repeat protein 14, and found up-regulated at all four time points. This study also reveals for the first time in a cnidarian that diverse G-protein coupled receptor (GPCR) homologs are highly regulated all along the infection and in response to LPS exposure (Supplementary Table 8). A variety of GPCRs are expressed in T cells and were recently linked to an important role in the mediation of immunity in humans (Mashaghi et al. 2016; Wang 2018; Lu and Cyster 2019). Thus Ep_GPCRs constitute fascinating molecular targets towards the identification of ancestral immune cells in early-diverging metazoans and/or the activation of innate immunity via sensory pathways.
Anthozoan innate immune response to pathogen infection
In the present study, we detected many more up-regulated DECs than down-regulated ones, but also the correlation between the stress intensity and the number of DECs was: the greater the stress over time, the greater the up-regulation (Table 1). Vidal-Dupiol and colleagues (2011a, b; 2014) have looked at the physiological and gene expression response of the coral Pocillopora damicornis (Pd) under temperature-dependent infection with Vc. Among the immune genes detected in Pd exposed to non-virulent Vc many also took part in this anemone’s response to Vp including NF-κB, AP-1, MyD88, and C3. However, these genes show the opposite regulation under the virulent Vc treatment, leading the authors to conclude the exact opposite to what was found in our study, “the greater the stress, the greater the down-regulation”. Therefore, besides the biological differences between anemones and corals, the confounding effects of temperature-induced bleaching and Vc’s virulence, the durational difference (i.e. days versus hours), as well as the various virulence mechanisms used by the different pathogens (Vc versus Vp), all contribute to the contradictory relationships found in Vidal-Dupiol et al. (2014) and our experiment.
Heat shock proteins (HSPs) are also among the actively regulated genes during a significant portion of the onset of Vp infection (Supplementary Table 4), which suggests that they play a role in defending against pathogenic invasion. This supports the findings in an experiment to determine the capacity of Ep for immunological priming and memory, in which Brown and Rodriguez-Lanetty (2015) used 2D fluorescent gel electrophoresis and mass spectrometry to detect differentially expressed HSPs in anemones exposed to sub-lethal Vc treatment for four weeks. Here, HSP70 and HSP90 DECs were up-regulated at 3, 6 and 12 hours in the response to Vp (Supplementary Table 4). Higher HSP expression levels in primed anemones were suggested to be an evolutionary trait that helps anemones to resist successive encounters with pathogens as long as they occur within a six-week period (Brown and Rodriguez-Lanetty 2015) and were thought to be more likely relevant to immunological priming conditions than pathogen clearance. However, the transcriptional changes of HSP transcripts observed here also suggest a role in the early defense against pathogens.
Our data provide strong support for the involvement of complement and coagulation cascades, NOD-like receptor signaling, lysosome, and apoptosis throughout the 12 hours of infection (Figure 2; Supplementary Table 6 and 7). These were also the pathways most affected in the responses of Ep to Vc detected using RNA-seq to explore the genetic links between anthozoan-algal symbiosis and innate immunity (Roesel and Vollmer 2019). Similar to our results, more DECs were found up-regulated than down-regulated in response to 24 hours exposure to Vc (i.e. 2,155 versus 1,552 DECs, respectively). Using KEGG pathway enrichment analysis, one pathway was significantly over-represented: the complement pathway under Vc treatment only. Our results build on Roesel and Vollmer’s (2019) results by pointing to the functional equivalence to canonical PRRs in cnidarians as well as many potential downstream immune signaling genes.