Antibacterial Mechanisms Of A Novel C-type Lectin In Eriocheir Sinensis

Chinese mitten crab(Eriocheir sinensis)is an important characteristic aquaculture species in China.It is plagued by bacterial and other diseases while creating considerable economic benefits,which has become one of the important bottlenecks restricting the healthy breeding and sustainable development of the crab.Invertebrates,including the crab,lack adaptive immunity and rely only on innate immunity to resist the invasion of pathogenic microorganisms.Therefore,in-depth analysis of the operation mechanism of innate immune response of E.sinensis will further enrich the understanding of the innate immune response system of invertebrates,provide a theoretical basis for disease prevention and control of E.sinensis and other crustaceans,and provide potential targets for disease resistance breeding of crustaceans.In this study,a novel C-type lectin(EsIgLectin)containing immunoglobulin(Ig)domain and C-type lectin-like domain(CTLD)was identified based on genome and transcriptome data of E.sinensis,and it was found that EsIgLectin was widely expressed in various tissues such as hemocytes.The m RNA and protein expression of EsIgLectin was significantly up-regulated in both hemocytes and intestine after abdominal or oral injection of Staphylococcus aureus and Vibrio parahaemolyticus,suggesting that EsIgLectin may play an important role in antibacterial immune response.In the study of Ig and CTLD domains,it was found that the two domains had synergistic effects in bacterial binding,promoting bacterial agglutination,inhibiting bacterial growth and bacterial clearance.Among them,the deletion of QPD motif in CTLD leads to a significant decrease in its ability to bind bacteria,promote bacterial agglutination,inhibit bacterial growth and bacteria clearance.Bioinformatics analysis showed that six cysteine residues in EsIgLectin could potentially produce three pairs of disulfide bonds.After treatment with chemical cross-linking agent BS3 and reduction and non-reduction protein loading buffers,SDS-PAGE and western blot detection showed that EsIgLectin could form EsIgLectin polymers through Ig domain.Further studies showed that recombinant proteins(r Es Ig_?Cys42,102?r EsIgLectin_?Cys42,102)with disulfide-deleted Ig domain could not form polymers,and could lead to extremely significant reduction in bacterial inhibition and clearance.The above results showed that CTLD and Ig domains played an important role in the antibacterial function of EsIgLectin.In order to study the potential of EsIgLectin as a secretory protein regulating cellular immunity through the surface receptor of hemocytes membrane,the binding and adhesion ability of recombinant proteins of EsIgLectin(r EsIgLectin,r Es Ig and r Es CTLD)to hemocytes were detected by laser confocal microscopy.The results showed that only Ig domain could bind and adhere to the surface of hemocytes membrane of E.sinensis.Further studies have found that in the recombinant proteins of the above domains,r EsIgLectin and r Es Ig have the ability to significantly promote the phagocytosis of E.sinensis hemocytes to bacteria,suggesting that Ig domain may have an interacting membrane surface receptor.Based on the screening of T7 phage library of E.sinensis hemocytes,it was found that r Es Ig may interact with poly immunoglobulin receptor(Esp IgR).Further GST pull-down assay not only confirmed the above protein interaction,but also clarified the mechanism of EsIgLectin Ig domain binding to Esp IgR extracellular Ig1 domain.On this basis,it was found that V.parahaemolyticus stimulation significantly up-regulated the m RNA level of Esp IgR gene in hemocytes,and the knockdown of Esp IgR in hemocytes could significantly offset the promotion of r EsIgLectin and r Es Ig on phagocytosis of hemocytes.The above results showed that EsIgLectin could regulate the phagocytosis of E.sinensis hemocytes by Esp IgR.Fluorescence in situ hybridization showed that EsIgLectin was expressed in intestinal epithelial cells,and the m RNA level of EsIgLectin was significantly up-regulated after oral injection of V.parahaemolyticus.In order to study the potential immune function of EsIgLectin in the intestine,E.sinensis were oral injected with r EsIgLectin,r Es Ig and r Es CTLD through mouth,and the 16S r DNA sequencing of intestinal contents was performed.The results showed that oral injected with r EsIgLectin,r Es Ig and r Es CTLD could change the?and?diversity and bacterial structure of the intestinal microbial of E.sinensis,so that the intestinal beneficial bacteria increased and the pathogenic bacteria decreased,thus regulating the intestinal microbial homeostasis.intestinal inflammation model and rescue model were established by oral injection of V.parahaemolyticus or r EsIgLectin,r Es Ig and r Es CTLD.In the rescue model,it was found that after injection of r EsIgLectin and r Es Ig,the density of intestinal epithelial cells increased,the number of epithelial cells dropped from the basement membrane decreased,and the intestinal wall became thicker,and other obvious intestinal inflammation remission phenomena were found.However,after injection of r Es CTLD,intestinal inflammation was not alleviated.The above results showed that EsIgLectin remission of intestinal inflammation was closely related to the Ig functional domain.The study also found that the m RNA level of Esp IgR was continuously up-regulated in intestine during the occurrence of intestinal inflammation,while the m RNA level of EsIgLectin was rapidly up-regulated in hemocytes in the late stage of intestinal inflammation.FITC-labeled V.parahaemolyticus was oral injected in the early and late stages of intestinal inflammation,respectively.It was found that only FITC signals could be detected in the hemolymph of the late stage of intestinal inflammation,indicating that V.parahaemolyticus could overflow from the intestine to the hemolymph in the late stage of intestinal inflammation to induce the up-regulation the m RNA level of EsIgLectin.Moreover,r EsIgLectin abdominal injection into the E.sinensis at the early and late stages of intestinal inflammation,it was found that EsIgLectin in the hemolymph at the late stage of intestinal inflammation could be transported to the intestine through Esp IgR to perform functions.After the establishment of EsIgLectin-Esp IgR axis,to study whether this axis has an impact on the survival of E.sinensis.In this paper,RNA interference technology was used to find that after knocking down EsIgLectin or Esp IgR,oral or abdominal injection of V.parahaemolyticus could lead to a significant increase in the mortality of E.sinensis.However,the injection of r EsIgLectin,r Es Ig or r Es CTLD and V.parahaemolyticus could reduce the mortality of E.sinensis,and the reduced mortality was dose-dependent on recombinant proteins.In addition,by knocking down Esp IgR first and then injecting r EsIgLectin and V.parahaemolyticus,it was found that EsIgLectin affected the mortality of E.sinensis in an Esp IgR-dependent manner.To sum up,this study reveals that EsIgLectin regulates the antibacterial immunity of E.sinensis through intestine-hemocytes axis,which not only enriches the innate immune theory of crustaceans,but also provides a new idea for the prevention and control of crustacean diseases such as shrimp and crab.