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Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

Recently, receptor interacting protein (RIP)-1 has been recognized as an intracellular sensor at the crossroads of apoptosis, necroptosis, and cell survival. To reveal when this crucial molecule originated and how its function in integrating stress signals evolved, in this study we report on two RIP1 homologs in Chinese amphioxus (Branchiostoma belcheri tsingtauense), designated B. belcheri tsingtauense RIP1a and B. belcheri tsingtauense RIP1b. Phylogenetic analysis indicates that they are generated by domain recombination and lineage-specific duplication. Similar to human RIP1, both B. belcheri tsingtauense RIP1a and B. belcheri tsingtauense RIP1b activate NF-κB in a kinase activity-independent manner and induce apoptosis through the Fas-associated death domain protein-caspase cascade. Moreover, we found that the natural point mutation of Q to I in the RIP homotypic interaction motif of B. belcheri tsingtauense RIP1a provides negative feedback for amphioxus RIP1-mediated signaling. Thus, our study not only suggests that RIP1 has emerged as a molecular switch in triggering cell death or survival in a basal chordate, but also adds new insights into the regulation mechanisms of RIP1-related signaling, providing a novel perspective on human diseases mediated by RIP1.

It has been speculated that before vertebrates evolved somatic diversity-based adaptive immunity, the germline-encoded diversity of innate immunity may have been more developed. Amphioxus occupies the basal position of the chordate phylum and hence is an important reference to the evolution of vertebrate immunity. Here we report the first comprehensive genomic survey of the immune gene repertoire of the amphioxus Branchiostoma floridae. It has been reported that the purple sea urchin has a vastly expanded innate receptor repertoire not previously seen in other species, which includes 222 toll-like receptors (TLRs), 203 NOD/NALP-like receptors (NLRs), and 218 scavenger receptors (SRs). We discovered that the amphioxus genome contains comparable expansion with 71 TLR gene models, 118 NLR models, and 270 SR models. Amphioxus also expands other receptor-like families, including 1215 C-type lectin models, 240 LRR and IGcam-containing models, 1363 other LRR-containing models, 75 C1q-like models, 98 ficolin-like models, and hundreds of models containing complement-related domains. The expansion is not restricted to receptors but is likely to extend to intermediate signal transducers because there are 58 TIR adapter-like models, 36 TRAF models, 44 initiator caspase models, and 541 death-fold domain-containing models in the genome. Amphioxus also has a sophisticated TNF system and a complicated complement system not previously seen in other invertebrates. Besides the increase of gene number, domain combinations of immune proteins are also increased. Altogether, this survey suggests that the amphioxus, a species without vertebrate-type adaptive immunity, holds extraordinary innate complexity and diversity.

A novel F4-carbohydrate recognition domain (CRD) – linker –F3-CRD-type bi-CRD Branchiostoma belcheri tsingtauense galectin (BbtGal)-L together with its alternatively spliced mono-CRD isoform BbtGal-S from amphioxus intestine was encoded by a 9488-bp unique gene with eight exons and seven introns. The recombinant proteins of BbtGal were found to have b-galactosidebinding activity, indicating that BbtGal was a member of the galectin family. Phylogenetic analysis of this gene along with its splicing form and genome structure suggested that the BbtGal gene was the primitive form of the chordate galectin family. Real-time polymerase chain reaction analyses (PCR) indicated that BbtGal mRNA was expressed during all stages of embryonic development. In terms of tissue distribution, BbtGal-L mRNA was mainly expressed in the immunity-related organs, such as hepatic diverticulum, intestine, and gill, but BbtGal-S was ubiquitously expressed in all tissues. The expression of BbtGal-L mRNA was elevated after acute challenge with various microorganisms, but BbtGal-L only bound to specific bacteria. The immune function of BbtGal was consistent with its localization both outside and inside the cell. Our study on amphioxus galectin may help further understanding of the evolution of chordate galectin in terms of host – pathogen interaction in the immune system.

Two novel tumor necrosis factor receptors, Bbt-TNFR1 and Bbt-TNFR2, were isolated from Chinese amphioxus, the closest relative to vertebrate. The mRNA of Bbt-TNFR1 encoded a type I membrane protein of 452 amino acids, including four cysteine-rich domains in the extracellular region and a putative TRAF6-binding site at its 154aa long cytoplasmic tail. Bbt-TNFR2 was a 304aa long type I membrane protein, featuring three cysteine-rich domains and a short cytoplasmic tail of just 13 amino acids. Southern blot revealed that Bbt-TNFR1 was a single copy gene, while Bbt-TNFR2 was presented in multiple copies. Sequence comparison indicated that both Bbt-TNFR1 and Bbt-TNFR2 were weakly similar to LT-bR, HVEM, TNFR2, CD40, OX40 and DcR3. Real-time PCR showed that Bbt-TNFR1 and Bbt-TNFR2 were regulated during development and finally had high expression in mucosa-rich tissues in adult stage. Furthermore, up-regulated expression of both genes was also observed in guts after Gram-positive bacteria challenge. However, not like Bbt-TNFR2's slowly and gradually augmentation in the following 48 h, expression of Bbt-TNFR1 dramatically surged up within 4 h and then subsided rapidly. Taking together, Bbt-TNFR1 and Bbt-TNFR2 may involve in the host defense of Chinese amphioxus via distinct fashions.

Recently, amphioxus has served as a model for studying the origin and evolution of vertebrate immunity. However, little is known about how microRNAs (miRNAs) are involved in the immune defense in amphioxus. In this article, we present a systematic study of amphioxus miRNAs in the acute-phase response to bacterial infection; miR-92d was found to regulate the complement pathway in this basal chordate. We identified all 155 possible miRNAs present in the amphioxus Branchiostoma belcheri genome by bioinformatics analyses, including 57 newly identified miRNAs (called bbe-miRNAs), and characterized the miRNA expression pattern. Four miRNAs (bbe-miR-7, bbe-miR-4868a, bbe-miR-2065, and bbe-miR-34b) were upregulated and bbe-miR-92d was downregulated under the challenge of both Vibrio anguillarum and Staphylococcus aureus bacteria. We further predicted miRNA targets and identified mRNA targets of immune-related miRNA using the hybrid PCR method. We propose that miR-92d regulates the complement pathway through targeting C3 for controlling the acute immune response to bacterial infections. This study provides evidence for the complex immune regulation of miRNAs in the acute-phase response in basal chordates.

The ficolin-mediated complement pathway plays an important role in vertebrate immunity, but it is not clear whether this pathway exists in invertebrates. Here we identified homologs of ficolin pathway components from the cephalochordate amphioxus and investigated whether they had been co-opted into a functional ficolin pathway. Four of these homologs, ficolin FCN1, serine protease MASP1 and MASP3, and complement component C3, were highly expressed in mucosal tissues and gonads, and were significantly up-regulated following bacterial infection. Recombinant FCN1 could induce hemagglutination, discriminate among sugar components, and specifically recognize and aggregate several bacteria (especially Gram-positive strains) without showing bactericidal activity. This suggested that FCN1 is a dedicated pattern-recognition receptor. Recombinant serine protease MASP1/3 formed complexes with recombinant FCN1 and facilitated the activation of native C3 protein in amphioxus humoral fluid, in which C3 acted as an immune effector. We conclude that amphioxus have developed a functional ficolin-complement pathway. Because ficolin pathway components have not been reported in non-chordate species, our findings supported the idea that this pathway may represent a chordate-specific innovation in the evolution of the complement system.

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and adaptive immunities. Its globular signature domain is also found in a variety of noncomplement protein that can be grouped together as a C1q family. In this study, we have cloned and identified a novel C1q family member in cephalochordate amphioxus and named it as AmphiC1q1. The high transcriptional levels of this gene were detected during all stages of embryonic development, and the section in situ hybridization demonstrated that AmphiC1q1 was mainly expressed in the ovary, intestine, and nerve system of mature individuals. The transcript of AmphiC1q1 was up-regulated by LPS and Gram-negative bacteria, but hardly by lipoteichoic acid and Staphylococcus aureus. The recombinant AmphiC1q1 protein could not bind with N-acetyl-glucosamine and did not possess hemagglutinating activity, indicating that AmphiC1q1 could not act as its lamprey homologue. But both the full-length protein and its truncated globular domain of C1q protein could interact with LPS. Moreover, recombinant AmphiC1q1 protein could inhibit collagen-induced platelet aggregation, but the truncated globular C1q domain protein would not, indicating that the blocking activity of AmphiC1q1 protein was via the collagen region of the protein. Our study on the primitive form of C1q family in protochordate will shed a light on understanding the gradual functional evolution of C1q family and eventual formation of mammalian homologues.

To investigate the evolution and immune function of C-type lectin in amphioxus, the primitive representative of the chordate phylum, we identified three C-type lectins consisting solely of a carbohydrate recognition domain and N-terminal signal peptide and found that they had distinct express patterns in special tissues and immune response to stimulations analyzed by quantitative real-time PCR. We characterized the biochemical and biological properties of AmphiCTL1, which was dramatically up-regulated in amphioxus challenged with Staphylococcus aureus, Saccharomyces cerevisiae, and zymosan. Immunohistochemistry demonstrated that the localization of AmphiCTL1 protein was exclusively detected in the inner folding tissues of the hepatic diverticulum. Recombinant AmphiCTL1 was characterized as a typical Ca2+-dependent carbohydrate-binding protein possessing hemagglutinating activity, preferentially bound to all examined four Gram-positive bacteria and two yeast strains, but had little binding activity toward four Gram-negative bacteria we tested. It aggregated S. aureus and S. cerevisiae in a Ca2+-dependent manner and specifically bound to insoluble peptidoglycan and glucan, but not to LPS, lipoteichoic acid, and mannan. Calcium increased the intensity of the interaction between AmphiCTL1 and those components, but was not essential. This lectin directly killed S. aureus and S. cerevisiae in a Ca2+-independent fashion, and its binding to microorganism cell wall polysaccharides such as peptidoglycan and glucan preceded microbial killing activity. These findings suggested that AmphiCTL1 acted as a direct microbial killing C-type lectin through binding microbial targets via interaction with peptidoglycan and glucan. Thus, AmphiCTL1 may be an evolutionarily primitive form of antimicrobial protein involved in lectin-mediated innate immunity.