The Anti-microbial Functions Of Immune Effectors In The Hemocytes Of Chinese Mitten Crab, Eriocheir Sinensis

The Chinese mitten crab(Eriocheir sinensis) belongs to the Arthropoda, Crustacea and Decapoda, which is one of the most important aquaculture species and widely cultivated in Southeast Asia. However, because of the intensive aquaculture, frequent outbreaks of diseases due to the infection of bacteria, virus and rickettsia have caused decreased production and catastrophic economic losses in the past decade. Anyway, understanding the immune system and host defense mechanism is the basis for disease resistance and drugs developping. Therefore, it is necessary to study the innate immune mechanisms of E. sinensis. Unlike vertebrates, invertebrates do not have a truly adaptive immunity generated on the basis of memory and targeted immunoglobulin production. Instead, they have an efficient innate immune system to defend against invading foreign materials. The defense system of invertebrates is based on both cellular and humoral immune responses. In these processes, conserved pathogen-associated molecular patterns (PAMPs), which including peptidoglycans (PGN), lipopolysaccharides (LPS) and β-1,3-glucans in microorganisms that invade invertebrate hosts can be recognize by a variety of pattern recognition receptors (PRRs) either free in plasma or associated with immune cells and activating cellular signaling pathway. Then regulate the expression of antimicrobial peptides.In order to advance immune mechanism research, a modified primary cell culture method for hemocytes from E. sinensiswas established using L-15 medium lacking FBS and CO2 at 28℃. Under these conditions, cultured cells were stably propagated for 30 days. To evaluate cellular activity, expression levels of certain critical immune-related genes, melanization of the hemocytes, and the phosphorylation status of key molecules in cell signaling pathways were analyzed. mRNA expression level of EsDWD1, EsLecG and EsToll2 were upregulated after LPS, PGN and GLU stimulation. Meanwhile, stimulated by Vibrio parahemolyticus led to the melanization of the hemocytes, and the melanization was more obviously with the increasing concentration of bacterias. Moreover, exposure to V. parahemolyticus significantly induced JNK, ERK and p38 phosphorylation, with the greatest extent of phosphorylation observed 30 min post-induction. Collectively, our results establish a technique for E. sinensis hemocyte primary culture and demonstrate appropriate immunological response, regulation and activity.In order to understanding the diversity extracellular proteins releaesd from hemocytes, which were stimulated by different microorganism, based on the primary culture of hemocytes, the medium was investigated using isobaric tags for relative and absolute quantitation (iTRAQ) after Vibrio parahemolyticus (gram-negative) and Staphylococcus aureus (gram-positive) stimulation respectively. Then, in the results, we screened several immune effectors increasing significantly after bacterial stimulation, which including transglutaminase (TGase) and C-type lectins (CTL, CTLD cintaining protein). In the following study, the immune functions of these two proteins were studied thoroughly.Transglutaminase (TGase) is critical for blood coagulation, a conserved immunological defense mechanism among invertebrates. Here, a 3248-bp (full-length) TGase cDNA in Eriocheir sinensis (EsTGase) was cloned, with a 2274-bp open reading frame (ORF) encoding a 757 amino acid protein containing two transglut domains, one TGase/protease-like homolog domain and a KGD (Lys-Gly-Asp) motif. Phylogenetic analysis demonstrated that EsTGase appeared earlier in evolution compared with TGases of other crustaceans and mammals. EsTGase mRNA was mainly detected in hemocytes and up-regulated post-challenge with bacteria (Vibrio parahemolyticus and Staphylococcus aureus), suggesting an immune function for this gene. Moreover, the EsTGase activity in hemocytes challenged with V. parahaemolyticus and S. aureus was decreased significantly. RNA interference of EsTGase down-regulating expression of immune-related genes CrusEs2, EsLecG and Es-DWD1 with or without bacteria stimulation in vitro. Furthermore, absence of EsTGase led to higher bacterial counts in the hemocyte culture medium. Thus, EsTGase is an important component of the crab immune response and is involved in the regulation of certain immune-related genes, particularly those encoding anti-microbial peptides.C-type lectins (CTLs) are pattern recognition proteins that play significant roles in the innate immune system by identifying and eliminating pathogens. Here, we have reported a CTL (EsLecH) from the Chinese mitten crab that can bind to microorganisms and regulate antimicrobial peptide (AMP) expression via the c-Jun N-terminal kinase (JNK) pathway. EsLecH was found to have an N-terminal signal peptide and a single carbohydrate recognition domain. The EsLecH transcript was detected abundantly in various tissues, and it was significantly upregulated in hemocytes after challenging with lipopolysaccharides and bacteria. Recombinant (r)EsLecH could bind to microorganisms, but at different levels. Ca2+ significantly increased rEsLecH binding affinity to microorganisms. Furthermore, growth inhibition by rEsLecH increased with increasing rEsLecH levels. Knockdown of EsLecH was accompanied by a significant reduction in AMP expression and JNK phosphorylation; AMP expression was reduced with JNK silencing and can not rescued by rEsLecH when absence of JNK. These results indicate that EsLecH could regulate AMPs via JNK signaling.