Characterization and comparative analyses of two amphioxus intelectins involved in the innate immune response.

Intelectin is a new type of soluble galactofuranose-binding lectin involved in innate immunity. Here we report another intelectin homolog, AmphiITLN239631, obtained from amphioxus, the transitional form between vertebrates and invertebrates. AmphiITLN239631 encoded 396 amino acids with a highly conserved fibrinogen-related domain (FReD), An intelectin domain and a putative Collagen domain. AmphiITLN239631 was ubiquitously expressed in all tissues we tested and transcripts in skin increased after challenge of both Escherichia coli and Staphylococcus aureus, although in different levels. Recombinant AmphiITLN239631 expressed in E. coli system could agglutinate both Gram-positive and Gram-negative bacteria in a calcium independent manner. Furthermore, recombinant protein was able to bind to lipopolysaccharide (LPS) and peptidoglycan (PGN), the major components of Gram-positive and Gram-negative bacteria cell walls, respectively. We also compared AmphiITLN239631 with previously identified AmphiITLN71469 and found that their tissue specificities, expression patterns upon bacteria challenge, and polysaccharide-binding affinities etc vary considerably. Our results could provide insight into the evolution and function of the intelectin family.

Genome-wide analyses of amphioxus microRNAs reveal an immune regulation via miR-92d targeting C3.

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.

Amphioxus CaVPT and creatine kinase are crucial immune-related molecules in response to bacterial infection and immunization.

Although a great progress has been made, our understanding of innate immunity is incomplete. Here, we hypothesize that the innate immune response to pathogens is attributed into a group of functional proteins. The group contains information on host status post bacterial entry (infection or immunity) and bacterial species (Gram-positive or Gram-negative bacteria). Investigation of the group of proteins may result in disclosing of biomarkers identifying the status and species. For this regard, differential proteomics approach coupled with the pattern recognition methods are used to identify biomarkers from the proteins that being specifically regulated during the innate immune response of amphioxus to Gram-positive and Gram-negative bacteria with live or dead status. Four proteins, Calcium vector protein (CaVP), sarcoplasmic calcium-binding protein (SCP), CaVP-target protein (CaVPT) and creatine kinase (CK), are selected as the key biomarkers. Since immunoprotection of CaVP and SCP has been reported, the role of CaVPT and CK are further investigated. Gut CaVPT appears in dying amphioxus, whereas humoral fluid CK downregulates and gut CK keep no change in animals with immunity. The responses are stronger in Gram-negative than Gram-positive bacteria. These results indicate that CaVPT, CK, CaVP and SCP are the most important biomarkers to uncover amphioxus innate immunity to bacteria, and the approach is an efficient way to identify key biomarkers.

A novel short peptidoglycan recognition protein in amphioxus: identification, expression and bioactivity.

Peptidoglycan recognition proteins (PGRPs) are widely distributed in invertebrates and vertebrates, and structure-activity relationship of insect and mammalian PGRPs has been well characterized, but functional and structural insights into PGRPs in other species are rather limited. Here we identified a novel short PGRP gene from the amphioxus Branchiostoma japonicum, named pgrp-s, which possesses a domain combination of ChtBD1 domain-PGRP domain, which is unique to all known PGRPs. Amphioxus pgrp-s was predominantly expressed in the hepatic caecum, hind-gut and muscle in a tissue-specific manner. Recombinant PGRP-S, rPGRP-S, and truncated protein with ChtBD1 domain deleted, rP86/250, both showed affinity to Dap-type PGN, Lys-type PGN and chitin. Consistently, they were also able to bind to Escherichia coli, Staphylococcus aureus and Pichia pastoris. Moreover, both rPGRP-S and rP86/250 had amidase enzymatic activity, capable of hydrolyzing Dap-type and Lys-type PGNs. Like vertebrate PGRPs, rPGRP-S was directly microbicidal, capable of killing E. coli, S. aureus and P. pastoris, whereas rP86/250 only inhibited the growth of E. coli and S. aureus, and its anti-P. pastoris activity was significantly reduced. It is clear that neither the binding of amphioxus PGRP-S nor its amidase enzymatic activity depend on the N-terminal ChtBD1 domain, but its antifungal activity does. Collectively, these data suggested that amphioxus PGRP-S may function as a multivalent pattern recognition receptor, capable of recognizing PGN and chitin, a microbicidal agent, capable of killing bacteria such as E. coli and S. aureus and fungus like P. pastoris, and probably a PGN scavenger, capable of hydrolyzing PGN.

Induction of phenoloxidase and other immunological activities in the humoral fluids of amphioxus Branchiostoma belcheri challenged with Lipopolysaccharide (LPS).

The knowledge concerning the humoral immunity is scarce in amphioxus Branchiostoma belcheri. This study measured the humoral parameters including phenoloxidase (PO) activity, lysozyme activity, antimicrobial activity, microbial agglutinin, and hemagglutinin in amphioxus humoral fluids before and after lipopolysaccharide (LPS) challenge. Humoral fluids from unchallenged Branchiostoma belcheri (B. belcheri) had PO activity, lysozyme, antimicrobial, microbial agglutinating, and hemagglutinating activities, which may represent part of the baseline level of innate immunity in this organism. After challenge with LPS, many humoral parameters were all increased significantly including the PO activity, lysozyme activity, growth-inhibiting activities against Gram-negative bacteria Escherichia coli (E. coli) and Vibrio alginolyticus (V. alginolyticus), growth-inhibiting activities against Gram-positive bacteria Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis), microbial agglutinating activities against Micrococcus lysodeikticus (M. lysodeikticus), B. subtilis, and S. aureus, and hemagglutinating activities against rabbit and human A and O erythrocytes. In contrast, the agglutinating activities against V. harveyi and E. coli and the hemagglutinating activity against human B erythrocytes in the humoral fluids were reduced in response to LPS challenge. It appears that the humoral fluids of B. belcheri contain components that are able to differentiate different microbes and different human blood cell types.

Identification and characterization of an amphioxus matrix metalloproteinase homolog BbMMPL2 responding to bacteria challenge.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases mainly involved in extracellular matrix (ECM) degradation. We have cloned and identified BbMMPL2 as homolog of MMPs from adult amphioxus. Recombinant BbMMPL2 proteins underwent self-processing during refolding in vitro. The final ~23 kDa polypeptide displayed proteolytic activity against ECM components like casein, gelatin, collagen IV and fibrinogen, but not laminin, fibronectin or α1-PI. This activity could be inhibited by GM6001 and TIMP-1/2. In addition, real-time RT-PCR analysis revealed that BbMMPL2 expressed in all issues/organs in adult amphioxus we tested. Its transcription was significantly up-regulated 12 h post immune challenge by Escherichia coli in epidermis and hepatic diverticulum but only slightly increased by Staphyloccocus aureus in epidermis. Furthermore, recombinant BbMMPL2-EGFP expressed in 293T and NIH/3T3 cells showed aggregation in cytoplasm and induced cell death. Our results provided new evidence that MMP was involved in immune response which could be conserved through evolution.

Characterization of the immune defense related tissues, cells, and genes in amphioxus.

Amphioxus is an important animal model for phylogenetic analysis, including comparative immunology. Exploring the immune system in amphioxus contributes to our understanding of the origin and evolution of the vertebrate immune system. We investigated the amphioxus immune system using ultrastructural examination and in situ hybridization. The expression patterns of TLR1 (toll-like receptor 1), C1Q (complement component 1, q subcomponent), ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), SoxC, DDAHa (Dimethylarginine dimethylaminohydrolase a), and NOS (nitric oxide synthase) show that these genes play key roles in amphioxus immunity. Our results suggest that the epidermis and alimentary canal epithelium may play important roles in immune defense, while macrophages located in the coelom and so-called lymph spaces may also be crucial immune cells.

Functional characterization of a ficolin-mediated complement pathway in amphioxus.

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.

Gut CaVP is an innate immune protein against bacterial challenge in amphioxus Branchiostoma belcheri.

The importance of calcium-binding proteins in immune response of vertebrates is determined, but whether they have the role in invertebrates is largely unknown. In the present study, phylogenetic analysis indicated that calcium vector protein (CaVP), a protein unique to amphioxus, shared 68% similarity in amino acid sequence with human and mouse calmodulin (CaM). CaVP cDNA was cloned into a bacterial vector pET-32a, and its His-tagged fusion protein was produced in Eschherichia coli cells (BL21). The recombinant CaVP was purified by Ni-NTA column and SDS-PAGE, and then utilized for antibody preparing. The prepared antibodies could recognize amphioxus CaVP with high specificity. Further analysis by Western blotting showed that CaVP was detected in muscle and humoral fluid of normal animals and appeared in gut of bacterial immunized or challenged amphioxus. Interestingly, gut CaVP was significantly higher in a healthy sub-group than a wounded sub-group post bacterial challenge. This response was detected strongly in immunization and challenge by the same Gram-negative bacterium Vibro parahaemolyticus and weakly in immunization by V. parahaemolyticus and then challenge by Gram-negative Aeromonas hydrophila, whereas no any feedback was found in immunization by V. parahaemolyticus and challenge by Gram-positive Staphylococcus aureus. These findings indicate the importance of gut CaVP in response to bacterial challenge.

The evolution and regulation of the mucosal immune complexity in the basal chordate amphioxus.

Both amphioxus and the sea urchin encode a complex innate immune gene repertoire in their genomes, but the composition and mechanisms of their innate immune systems, as well as the fundamental differences between two systems, remain largely unexplored. In this study, we dissect the mucosal immune complexity of amphioxus into different evolutionary-functional modes and regulatory patterns by integrating information from phylogenetic inferences, genome-wide digital expression profiles, time course expression dynamics, and functional analyses. With these rich data, we reconstruct several major immune subsystems in amphioxus and analyze their regulation during mucosal infection. These include the TNF/IL-1R network, TLR and NLR networks, complement system, apoptosis network, oxidative pathways, and other effector genes (e.g., peptidoglycan recognition proteins, Gram-negative binding proteins, and chitin-binding proteins). We show that beneath the superficial similarity to that of the sea urchin, the amphioxus innate system, despite preserving critical invertebrate components, is more similar to that of the vertebrates in terms of composition, expression regulation, and functional strategies. For example, major effectors in amphioxus gut mucous tissue are the well-developed complement and oxidative-burst systems, and the signaling network in amphioxus seems to emphasize signal transduction/modulation more than initiation. In conclusion, we suggest that the innate immune systems of amphioxus and the sea urchin are strategically different, possibly representing two successful cases among many expanded immune systems that arose at the age of the Cambrian explosion. We further suggest that the vertebrate innate immune system should be derived from one of these expanded systems, most likely from the same one that was shared by amphioxus.

Gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri.

To understand the role of calcium-binding proteins of invertebrates in immunological response, amphioxus sarcoplasmic calcium-binding protein (SCP) was investigated in the present study. Following gene cloning, recombinant protein expression and purification and antibody preparation, the expression and alteration of SCP in the response to bacterial challenge were detected using Western blotting. SCP was not detected in the branchia, humoral fluid, gonad or in the gut of wounded animals, but it was abundant in muscle and appeared in the gut of healthy animals using Vibrio parahaemolyticus immunization and challenge. Furthermore, whether gut SCP possessed anamnestic response was investigated using cross-immune challenge between Gram-positive and -negative bacteria. Gut SCP showed stronger anamnestic activity or pattern-recognition in response to Gram-negative bacterium V. parahaemolyticus than Gram-positive bacterium Staphylococcus aureus. The response was faster and more species-specific to V. parahaemolyticus, whereas it was slower and longer to S. aureus. The reason why the response showed significant difference between Gram-positive and -negative bacteria awaits investigation. These results indicate that gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri.

A transferrin-like homolog in amphioxus Branchiostoma belcheri: Identification, expression and functional characterization.

Previous studies have shown the universal presence of transferrin (Tf) in both invertebrates and vertebrates, but little information is available regarding Tf in amphioxus, a protochordate on the evolutionary boundary between invertebrates and vertebrates. Here we isolated a Tf-like homolog from Branchiostoma belcheri, which encodes a deduced protein, BbTfl, of 1256 amino acids containing a N-terminal signal peptide, a conserved transferrin domain in its N-terminal lobe, with a putative iron-binding site consisting of Asp63 and Try188 and another transferrin domain in its C-terminal lobe with an long intervening sequence of 305 amino acids. Phylogenetic analysis shows BbTfl is grouped together with all the invertebrate Tfs and located at the base of melanotransferrins and other Tfs. Quantitative PCR analysis reveals that exposure to Escherichia coli and Vibrio anguillarum causes a significant increase in BbTfl expression mainly in the gut within 12-24h, suggesting that BbTfl is a positive acute phase reactant involved in the immune defense of B. belcheri. The recombinant N-terminal lobe, BbTflN, is able to bind iron and to inhibit E. coli and Staphylococcus aureus growth. Moreover, the antibacterial activity of BbTflN markedly decreases in the presence of excess iron. All these results provide a direct empirical evidence establishing a definitive link between binding to iron and bacterial growth-inhibiting activity. It is also shown that BbTfl is expressed in a tissue-specific manner, with the most abundant expression in the hepatic caecum, hind-gut and ovary, supporting the idea that the digestive system including the hepatic caecum of amphioxus is the primary tissue involved in acute phase response.

Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates.

About 500 million years ago, a new type of adaptive immune defense emerged in basal jawed vertebrates, accompanied by morphological innovations, including the thymus. Did these evolutionary novelties arise de novo or from elaboration of ancient genetic networks? We reconstructed the genetic changes underlying thymopoiesis by comparative genome and expression analyses in chordates and basal vertebrates. The derived models of genetic networks were experimentally verified in bony fishes. Ancestral networks defining circumscribed regions of the pharyngeal epithelium of jawless vertebrates expanded in cartilaginous fishes to incorporate novel genes, notably those encoding chemokines. Correspondingly, novel networks evolved in lymphocytes of jawed vertebrates to control the expression of additional chemokine receptors. These complementary changes enabled unprecedented Delta/Notch signaling between pharyngeal epithelium and lymphoid cells that was exploited for specification to the T cell lineage. Our results provide a framework elucidating the evolution of key features of the adaptive immune system in jawed vertebrates.

An amphioxus TLR with dynamic embryonic expression pattern responses to pathogens and activates NF-kappaB pathway via MyD88.

A big bang expansion of the Vertebrate-type (V-type) TLRs was reported in amphioxus. To shed lights on its implications, a unique TLR which is reversely inserted into an intron of amphioxus PSMB7-10 by retrotransposition in the highly polymorphic proto-MHC region was cloned from Chinese amphioxus (Branchiostoma belcheri tsingtauense) and named as bbtTLR1. In situ assays showed that bbtTLR1 was predominantly expressed in pharynx and gut from larva to adult stages, which are considered as the first frontlines of amphioxus defense system. Acute immune challenges revealed that the expression of bbtTLR1 was stimulated by bacteria and their cell wall components, while suppressed by Glucan and Poly I:C in the digestive system. Amphioxus also had dozens of TIR adaptors from which we cloned bbtMyD88. BbtMyD88 expressed in 293T cells led to the activation of NF-kappaB pathway through its DEATH and middle domains. Moreover, this activation could be enhanced by bbtTLR1 through the direct association with bbtMyD88. In summary, this study provides evidence for the immune-relation of amphioxus V-type TLRs, and suggests that amphioxus TLR1 and MyD88 represent a basic evolutionary pathway.

The evolutionarily dynamic IFN-inducible GTPase proteins play conserved immune functions in vertebrates and cephalochordates.

Interferon (IFN)-inducible GTPases currently include four families of proteins: myxovirus resistant proteins (Mxs), guanylate-binding proteins (GBPs), immunity-related GTPase proteins (IRGs), and very large inducible GTPase proteins (VLIGs). They are all under conserved regulation by IFNs in humans and mice and play a critical role in preventing microbial infections. However, differences between vertebrates are poorly characterized, and their evolutionary origins have not been studied in detail. In this study, we performed comparative genomic analysis of the four families in 18 representative animals that yielded several unexpected results. Firstly, we found that Mx, GBP, and IRG protein families arose before the divergence of chordate subphyla, but VLIG emerged solely in vertebrates. Secondly, IRG, GBP, and VLIG families have experienced a high rate of gene gain and loss during the evolution, with the GBP family being lost entirely in two pufferfish and VLIG family lost in primates and carnivores. Thirdly, the regulation of these genes by IFNs is highly conserved throughout vertebrates although the VLIG protein sequences in fish have lost the first 870 amino acid residues. Finally, amphioxus IFN-inducible GTPase genes are all highly expressed in immune-related organs such as gill, liver, and intestine and are upregulated after challenge with PolyI:C and pathogens, although no IFNs or their receptors were detected in the current amphioxus genome database. These results suggest that IFN-inducible GTPase genes play conserved immune functions both in vertebrates and in cephalochordates.

EST analysis of the immune-relevant genes in Chinese amphioxus challenged with lipopolysaccharide.

It is generally accepted that the adaptive immune system is only present in vertebrates but not in invertebrates. Amphioxus is the most basal chordate and hence is an important reference to the evolution of the adaptive immune system. Here, a cDNA library of lipopolysaccharide-challenged amphioxus was constructed in order to identify immune genes. A total of 3024 expressed sequence tags (ESTs) were examined and 63 out of 398 annotated genes (16.3%) appeared related to immunity. Most of them encode cell adhesion molecules or signal proteins that are involved in immune responses. Although the key molecules such as TCR, MHC, Ig or VLR involved in the adaptive immune system were not identified in our database, we demonstrated the presence of histocompatibility-relevant genes and lymphocyte immune signaling-relevant genes. These findings support the statement that amphioxus presents some components that may be recruited by adaptive immune processes.

Induction of phenoloxidases in the humoral fluids of amphioxus Branchiostoma belcheri by Vibrio alginolyticus and Escherichia coli.

The humoral immune responses of amphioxus Branchiostoma belcheri to microbial challenge remain open to date. Here we examined the changes in PO activity in the humoral fluids in amphioxus before and after challenge with Escherichia coli and Vibrio alginolyticus. It was found that PO activity in the humoral fluids is markedly increased by challenge with E. coli and V. alginolyticus; and the microbial challenge results in a significant rise in subunit 2 of the three PO subunits, making PO subunit 2 a marker enzyme responsive to microbial challenge. This is the first report on microbial induction of the immune-related molecules like PO in B. belcheri.

Responses of alternative complement expression to challenge with different combinations of Vibrio anguillarum, Escherichia coli and Staphylococcus aureus: evidence for specific immune priming in amphioxus Branchiostoma belcheri.

The existence of specific immunological priming in protection upon secondary exposure in invertebrates remains controversial. By exploring the changes in the expression patterns of Bf, C3 and C6, key genes involved in the alternative complement pathway (AP), after challenge with different combinations of three bacteria Vibrio anguillarum, Escherichia coli and Staphylococcus aureus, we show that re-exposure to the same species of bacteria or the different species of the same (Gram-negative or Gram-positive) class of bacteria results in a significant increase in the expression of Bf, C3 and C6, and an earlier occurrence of gene expression peak compared with the first exposure in amphioxus Branchiostoma belcheri; in contrast, re-exposure to the different class of bacteria did not induce such responses. These characteristics appear to bear some analogy to the immunological memory in vertebrates, suggesting that amphioxus B. belcheri possesses the ability to discriminate between classes of microorganisms. Moreover, our results for the first time establish a link between the alternative complement components and the specific immune priming.

Novel genes dramatically alter regulatory network topology in amphioxus.

BACKGROUND: Regulation in protein networks often utilizes specialized domains that 'join' (or 'connect') the network through specific protein-protein interactions. The innate immune system, which provides a first and, in many species, the only line of defense against microbial and viral pathogens, is regulated in this way. Amphioxus (Branchiostoma floridae), whose genome was recently sequenced, occupies a unique position in the evolution of innate immunity, having diverged within the chordate lineage prior to the emergence of the adaptive immune system in vertebrates. RESULTS: The repertoire of several families of innate immunity proteins is expanded in amphioxus compared to both vertebrates and protostome invertebrates. Part of this expansion consists of genes encoding proteins with unusual domain architectures, which often contain both upstream receptor and downstream activator domains, suggesting a potential role for direct connections (shortcuts) that bypass usual signal transduction pathways. CONCLUSION: Domain rearrangements can potentially alter the topology of protein-protein interaction (and regulatory) networks. The extent of such arrangements in the innate immune network of amphioxus suggests that domain shuffling, which is an important mechanism in the evolution of multidomain proteins, has also shaped the development of immune systems.

Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity.

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.