The Roles Of Pattern Recognition Receptors In Eriocheir Sinensis Specific Innate Immunity

The freshwater Chinese mitten crab, Eriocheir sinensis (Henri Milne Edwards1854), is a traditional savory food especially in the Yangtze River Area, and comprises one of the most economically important freshwater aquatic species of China. With the development of intensive aquaculture, various diseases caused by bacteria, viruses or rickettsia-like organisms have also begun to emerge and have resulted in enormous economic loss. Therefore, an improved understanding of the innate immunity of this particular crab and its bio-defense mechanisms has become a research priority. In the absence of an adaptive immune system, which in the higher vertebrates is characterized by the vast diversity of somatically rearranged immunological receptors like antibodies, invertebrates including E. sinensis rely solely on innate immune mechanism to alleviate the long lasting selective pressure from various kinds of parasites. The most straightforward basis for immune specificity in these organisms relies on the genetic diversity of pattern recognition receptors (PRRs) and/or immune effectors. In my PhD project, I firstly constructed comparative transcriptomic libraries using Next Generation Sequencing approach (e.g. via Roche454and Illumina), subsequently carried out sequence alignments and annotations. Aiming to dissect the molecular mechanisms of invertebrate specific immune responses, I focused on the function of some important PRRs in crustaceans such as the C-type lectin domain-(CTLD) containing proteins which were identified to be abundant in immune organs or cells like the hepatopancreas or hemocytes with highly molecular diversity (40isoforms at least). I also studied other pattern recognition molecules such as Dscam, which was detected to have surprisingly molecular diversity using exons alternative splicing, acted as important role in the innate immune system of E.sinensis.As an important member of PRRs, CTLD containing proteins exist as either transmembrane receptors or soluble proteins in circulating fluids. They play crucial roles in innate immunity such as non-self-recognition and clearance of invading microorganisms, via recognizing and non-covalently binding to specific sugar moieties and agglutinating pathogens by binding to cell surface glycoproteins and glycoconjugates. Based on our previously performed transcriptomic analysis using large-scale RNA-Seq (plus unpublished data from differentially expression transcriptomes from hemocytes), we successively obtained numerous sequences with identical CTLDs inclusions (approximately40unique sequences). Subsequently, we performed a set of in-vivo immuno-challenge tests of these CTLDs (to be continued) using Lipopolysaccharide (LPS:an endotoxin from Gram-negative bacterial Escherichia coli) as a stimulus. Real-time quantitative PCR results revealed that:i) two of these CTLDs (EsLecA and EsLecG) were immune-inducible in all tested immune organs/cells such as hepatopancreas, gills and hemocytes; and ii) an additional CTLD (EsLecF) was immune-inducible in hepatopancreas only. To dissect their exact roles in innate immunity, we performed a series of in-vitro tests with the respective recombinant CTLD proteins, which were generated using prokaryotic expression systems combined with affinity chromatography. The in-vitro protein-level analysis demonstrated that, these selected three CTLD proteins consistently expressed wide spectrum microbial binding activities and could all differentially induced microbe aggregation. Surprisingly, their microbial-binding and microbial-aggregation activities were calcium-independent. Moreover, microbial growth inhibitory and also antibacterial activity assays revealed the proteins’abilities of suppressing microbial growth and directly killing microbes respectively. Furthermore, an encapsulation assay demonstrated an opsonic function of these CTLD proteins.As discussed above, invertebrates lack adaptive immunity which can mediate both immune specificity and memory. At the same time, invertebrate taxa are known from phenotypic analysis to express high levels of immune specificity which possibly depends on some diversified protein family involved in pathogen recognition or elimination. The Dscam proteins (Down syndrome cell adhesion molecule) belong to the immunoglobulin super family (IgSF) and are of particular interest in this context because of their diversified domain architecture. We were able to successfully isolate and characterize the first crab Dscam from E. sinensis. EsDscam shows the typical Dscam domain architecture, including one signal-peptide,10immunoglobulin (Ig) domains,6fibronectin type III domains (FNIII), one transmembrane domain (TM) and one cytoplasmic tail. It was detected to have four hypervariable regions in the N-terminal halves of Ig2(25) and Ig3domain (30), the complete Ig7(18) and also the transmembrane domain (2), thus potentially generating at least27,000unique isoforms through exons alternative splicing mechanisms. Transcription of EsDscam was both i) detected in all tissues, especially those of the immune system, digestive system and nerve system; and ii) significantly induced in hemocytes upon exposure to pathogen surface molecules, e.g. LPS, peptidoglycans (PGN) and β-1,3-glucans (Glu) injection. Importantly, both membrane-bound and secreted Dscam isoforms were expressed in E. sinensis. Moreover, the secreted isoforms were extensively transcribed upon challenge with different pathogen molecules. Results from immuno-localization assay revealed that EsDscam evenly distributed on the cell surface of hemocytes. These findings strongly suggest that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis.