The cyclic dinucleotide 2′3′-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis

Abstract

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2′3′ cyclic guanosine monophosphate-adenosine monophosphate (2′3′-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis. Using RNA sequencing, we found that 2′3′-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis. Many of the antiviral genes induced by 2′3′-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2′3′-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2′3′-cGAMP signaling in animals.

Starvation Decreases Immunity and Immune Regulatory Factor NF-κB in the Starlet Sea Anemone Nematostella vectensis

“…NF-κB is known for its role as master regulator of immunity 33,34 ; this role can include the regulation of other members in the NF-κB signaling pathway. One such member, TNF receptor-associated factor 3 ( TRAF3), h as been previously observed to be upregulated with NF-κB in Nv 25 . 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.…”

“…Pseudomonas aeruginosa is a Gram-negative bacterium that is pathogenic in a variety of hosts including some plants, invertebrates, and vertebrates 31,32 . Recently, P. aeruginosa strain PA14 was also shown to be pathogenic for juvenile Nv 25 . 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 × 10 8 CFU/ml of PA14.…”

“…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-fertilization 22,35 . Given the upregulation of NF-κB following infection with P. aeruginosa 25 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 (log 2 Fold Change = -0.96, raw p-value = 0.01), this trend was not significant after FDR correction.…”

“…Infection was performed essentially as described previously 25 . 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 OD 600 of ~0.1 in 1/3 ASW.…”

“…Transcription factor NF-κB (nuclear factor kappa B) has been intensively studied in animals from insects to vertebrates for its involvement in development and immunity 21 . Although the basic NF-κB pathway in Nv has been characterized 22 and has been linked to development 23 and immunity 24,25 , 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 immunity 21 .…”