Ancestral reconstruction reveals catalytic inactivation of activation-induced cytidine deaminase concomitant with cold water adaption in the Gadiformes bony fish.

BACKGROUND: Antibody affinity maturation in vertebrates requires the enzyme activation-induced cytidine deaminase (AID) which initiates secondary antibody diversification by mutating the immunoglobulin loci. AID-driven antibody diversification is conserved across jawed vertebrates since bony and cartilaginous fish. Two exceptions have recently been reported, the Pipefish and Anglerfish, in which the AID-encoding aicda gene has been lost. Both cases are associated with unusual reproductive behavior, including male pregnancy and sexual parasitism. Several cold water fish in the Atlantic cod (Gadinae) family carry an aicda gene that encodes for a full-length enzyme but lack affinity-matured antibodies and rely on antibodies of broad antigenic specificity. Hence, we examined the functionality of their AID. RESULTS: By combining genomics, transcriptomics, immune responsiveness, and functional enzymology of AID from 36 extant species, we demonstrate that AID of that Atlantic cod and related fish have extremely lethargic or no catalytic activity. Through ancestral reconstruction and functional enzymology of 71 AID enzymes, we show that this enzymatic inactivation likely took place relatively recently at the emergence of the true cod family (Gadidae) from their ancestral Gadiformes order. We show that this AID inactivation is not only concordant with the previously shown loss of key adaptive immune genes and expansion of innate and cell-based immune genes in the Gadiformes but is further reflected in the genomes of these fish in the form of loss of AID-favored sequence motifs in their immunoglobulin variable region genes. CONCLUSIONS: Recent demonstrations of the loss of the aicda gene in two fish species challenge the paradigm that AID-driven secondary antibody diversification is absolutely conserved in jawed vertebrates. These species have unusual reproductive behaviors forming an evolutionary pressure for a certain loss of immunity to avoid tissue rejection. We report here an instance of catalytic inactivation and functional loss of AID rather than gene loss in a conventionally reproducing vertebrate. Our data suggest that an expanded innate immunity, in addition to lower pathogenic pressures in a cold environment relieved the pressure to maintain robust secondary antibody diversification. We suggest that in this unique scenario, the AID-mediated collateral genome-wide damage would form an evolutionary pressure to lose AID function.

Genomic architecture of haddock (Melanogrammus aeglefinus) shows expansions of innate immune genes and short tandem repeats.

BACKGROUND: Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group. RESULTS: Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) - a relative of Atlantic cod - using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes. CONCLUSIONS: The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod - but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Innovation in Nucleotide-Binding Oligomerization-Like Receptor and Toll-Like Receptor Sensing Drives the Major Histocompatibility Complex-II Free Atlantic Cod Immune System.

The absence of MHC class II antigen presentation and multiple pathogen recognition receptors in the Atlantic cod has not impaired its immune response however how underlying mechanisms have adapted remains largely unknown. In this study, ex vivo cod macrophages were challenged with various bacterial and viral microbe-associated molecular patterns (MAMP) to identify major response pathways. Cytosolic MAMP-PRR pathways based upon the NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) were identified as the critical response pathways. Our analyses suggest that internalization of exogenous ligands through scavenger receptors drives both pathways activating transcription factors like NF-kB (Nuclear factor-kappa B) and interferon regulatory factors (IRFs). Further, ligand-dependent differential expression of a unique TLR25 isoform and multiple NLR paralogues suggests (sub)neofunctionalization toward specific immune defensive strategies. Our results further demonstrate that the unique immune system of the Atlantic cod provides an unprecedented opportunity to explore the evolutionary history of PRR-based signaling in vertebrate immunity.

The Genome of the Great Gerbil Reveals Species-Specific Duplication of an MHCII Gene.

The great gerbil (Rhombomys opimus) is a social rodent living in permanent, complex burrow systems distributed throughout Central Asia, where it serves as the main host of several important vector-borne infectious pathogens including the well-known plague bacterium (Yersinia pestis). Here, we present a continuous annotated genome assembly of the great gerbil, covering over 96% of the estimated 2.47-Gb genome. Taking advantage of the recent genome assemblies of the sand rat (Psammomys obesus) and the Mongolian gerbil (Meriones unguiculatus), comparative immunogenomic analyses reveal shared gene losses within TLR gene families (i.e., TLR8, TLR10, and the entire TLR11-subfamily) for Gerbillinae, accompanied with signs of diversifying selection of TLR7 and TLR9. Most notably, we find a great gerbil-specific duplication of the MHCII DRB locus. In silico analyses suggest that the duplicated gene provides high peptide binding affinity for Yersiniae epitopes as well as Leishmania and Leptospira epitopes, putatively leading to increased capability to withstand infections by these pathogens. Our study demonstrates the power of whole-genome sequencing combined with comparative genomic analyses to gain deeper insight into the immunogenomic landscape of the great gerbil and its close relatives.

Full characterization and transcript expression profiling of the interferon regulatory factor (IRF) gene family in Atlantic cod (Gadus morhua).

Atlantic cod (Gadus morhua) represents a unique immune system among teleost fish, making it a species of interest for immunological studies, and especially for investigating the evolutionary history of immune gene families. The interferon regulatory factor (IRF) gene family encodes transcription factors which function in the interferon pathway, but also in areas including leukocyte differentiation, cell growth, autoimmunity, and development. We previously characterized several IRF family members in Atlantic cod (Irf4a, Irf4b, Irf7, Irf8, and two Irf10 splice variants) at the cDNA and putative amino acid levels, and in the current study we took advantage of the new and improved Atlantic cod genome assembly in combination with rapid amplification of cDNA ends (RACE) to characterize the remaining family members (i.e. Irf3, Irf5, Irf6, Irf9, and two Irf2 splice variants). Real-time quantitative PCR (QPCR) was used to investigate constitutive expression of all IRF transcripts during embryonic development, suggesting several putative maternal transcripts, and potential stage-specific roles. QPCR studies also showed 11 of 13 transcripts were responsive to stimulation with poly(I:C), while 6 of 13 transcripts were responsive to lipopolysaccharide (LPS) in Atlantic cod head kidney macrophages, indicating roles for cod IRF family members in both antiviral and antibacterial responses. This study is the first to investigate expression of the complete IRF family in Atlantic cod, and suggests potential novel roles for several of these transcription factors within immunity as well as in early development of this species.

Successive Losses of Central Immune Genes Characterize the Gadiformes' Alternate Immunity.

Great genetic variability among teleost immunomes, with gene losses and expansions of central adaptive and innate components, has been discovered through genome sequencing over the last few years. Here, we demonstrate that the innate Myxovirus resistance gene (Mx) is lost from the ancestor of Gadiformes and the closely related Stylephorus chordatus, thus predating the loss of Major Histocompatibility Complex class II (MHCII) in Gadiformes. Although the functional implication of Mx loss is still unknown, we demonstrate that this loss is one of several ancient events appearing in successive order throughout the evolution of teleost immunity. In particular, we find that the loss of Toll-like receptor 5 predates the loss of Mx involving the entire Paracanthopterygii lineage. Using a time-calibrated phylogeny, we show that loss of MHCII and Mx overlap with major paleoclimatic and geological events indicating that these genetic changes were adaptive responses to the changing environment at the time.

Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity.