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.

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.

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.

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.

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.

Chlamydial symbionts in the enigmatic Xenoturbella (Deuterostomia).

Ultrastructural observations of the gastrodermal cells in the enigmatic Xenoturbella revealed numerous chlamydiae. They are related to "Candidatus Fritschea" and Simkania (Simkaniaceae) based on 16S and 23S rRNA. Their 23S rRNA gene contains an intron encoding a putative homing endonuclease. The chlamydiae were pleomorphic and formed intravacuolar colonies. They have flattened disk-shaped elementary bodies, either oval or bow tie-shaped in cross-section, and reticulate bodies that are spherical, polygonal or irregularly shaped. All stages have five-layered cell wall with rippled appearance. Bacteria were not observed in the nuclei. The association between the chlamydiae and Xenoturbella is characterized by absence of cytopathological effects; limited host cell response against the chlamydiae; the confinement of the chlamydiae to inclusions in some part of the host cell; and complete and uniform infection of all examined hosts.

Profile of acute immune response in Chinese amphioxus upon Staphylococcus aureus and Vibrio parahaemolyticus infection.

Amphioxus is traditionally considered as the living invertebrate most closely related to vertebrate. However, no systematic study was performed about how the amphioxus defends against the microbial invasion. Here we reported a profile of gene transcription after Staphylococcus aureus (S.c) and Vibrio parahaemolyticus (V.p) challenged by suppression subtractive hybridization (SSH). When compared with mammals, amphioxus has the same acute immune defense genes (lectins, metalloproteinase, lysozymes and antimicrobial peptide, etc.) as well as a similar pattern and level of temporal gene expression. In contrast, amphioxus was demonstrated to have some novel acute immune response genes in response to the microbial challenge, such as apextrin and dermatopontin, which have a 3500-fold and 900-fold induction after the V.p infection, respectively, suggesting new functions in early immune system for these two genes. Our results reported for the first time a profile of primitive immune system defense against infection in protochordate.

Agarivorans albus gen. nov., sp. nov., a gamma-proteobacterium isolated from marine animals.

Six bacterial strains were isolated from healthy marine organisms that were collected from the coast of the Kanto area in Japan. Phylogenetic analysis based on 16S rDNA sequence similarity showed that the six isolates formed a separate cluster in the gamma-Proteobacteria and were related to the genera Alteromonas and Glaciecola (<91.6 % similarity). The isolates were related closely to each other (DNA-DNA reassociation values of 74-93 %). The isolates had a polar flagellum and were Gram-negative, mesophilic, strictly aerobic rods that required salt for growth. Distinct phenotypic features of this group included the ability to hydrolyse agar and white pigmentation of colonies. The DNA G+C content of the isolates was 48-50 mol%. The major quinone was Q-8. Phenotypic characteristics of the isolates differed from those of members of the genera Alteromonas and GLACIECOLA: The name Agarivorans albus gen. nov., sp. nov. is proposed for the six isolates; the type strain is MKT 106(T) (=IAM 14998(T)=LMG 21761(T)).