CpG oligonucleotides bind TLR9 and RRM-Containing proteins in Atlantic Salmon (Salmo salar)

Abstract

BackgroundBacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates.ResultsIn the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes.ConclusionThe presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.

Insights into the Antiviral Immunity against Grass Carp (Ctenopharyngodon idella) Reovirus (GCRV) in Grass Carp

“…Fish TLR9 senses viral and bacterial DNA. The LRR sites in TLR9 molecule of teleost can sense a variety of CpG-oligodeoxynucleotides (CpG ODNs) motifs present in different bacteria [ 75 ]; Atlantic salmon ( Salmo salar ) TLR9 interacts with synthetic ODN via a CpG-independent but pH-dependent mechanism [ 76 ]; recent study highlights that TLR9 and TLR21 cooperatively mediate activity of CpG-ODNs in zebrafish [ 77 ].…”

Vaccine Adjuvants in Fish Vaccines Make a Difference: Comparing Three Adjuvants (Montanide ISA763A Oil, CpG/Poly I:C Combo and VHSV Glycoprotein) Alone or in Combination Formulated with an Inactivated Whole Salmonid Alphavirus Antigen

“…Among the best studied PRRs is the Toll-like receptor (TLR) family, of which 17 types have been described in different fish species [ 21 ]. The piscine TLRs includes TLR9 recognizing bacterial and viral DNA [ 22 , 23 ] and TLR3 and TLR22, which both recognize double-stranded (ds) RNA [ 24 , 25 ]. Studies by us and other groups have demonstrated that ligands for these receptors, such as synthetic CpG oligonucleotides (ODNs; TLR9 ligand) and polyI:C (TLR3/22 ligand) can stimulate the production of pro-inflammatory cytokines/chemokines and Type I IFNs, which increase the host’s ability to eliminate viral pathogens [ 26 , 27 ].…”

Host–parasite transcriptomics during immunostimulant-enhanced rejection of salmon lice (Lepeophtheirus salmonis) by Atlantic salmon (Salmo salar)

“…Although tlr9 was overexpressed in the group fed the immunostimulant diet, the expression of this gene decreased over time. Atlantic salmon CpG-ODN has been shown to bind tlr9 for signaling IFN-related genes (Iliev et al 2013); it is possible that the current immunostimulant is working through the same pathway, but this would require more work to confirm. Surprisingly, mmp9 was not increased in mRNA levels from the infection, as in many other studies, this gene is a reliable indicator of a response to lice infection.…”