Study On The Function Of Tetraspanin In Mitten Crabs During Spiroplasma Infection

Eriocheir sinensis is an important species in Chinese freshwater aquaculture industry and has important economic value.However,as the scale of aquaculture continues to expand,the diseases are becoming more and more serious.Among them,tremor disease(TD)is one of the most serious diseases of Eriocheir sinensis,which causing serious economic losses.A spiroplasma was identified as the pathogen of TD and named Spiroplasma eriocheiris.Previous studies have shown that the S.eriocheiris entered into the crab through the gill or surface and invaded its target cells,hemocytes.Then,through blood circulation,invading connective,nerve and other tissues caused the crab tremor.Tetraspanins belong to the transmembrane 4 superfamily(TM4SF),which are involved in basic cellular processes including cell growth,adhesion,differentiation,participated in recognition and infection of some pathogene and played an important role in the immune response.The differentially expressed proteins in the hemocytes of E.sinensis after S.eriocheiris infection were identified though iTraq technology.Among them,tetraspanin was down-regulated after infection with S.eriocheiris.Based on the previous research,in this study,firstly,obtained the full length of tetraspanin(EsTetraspanin)gene from Eriocheir sinensis by RACE technology,and then analyzed its sequence characteristics and expression patterns.Secondly,its role in the infection of S.eriocheiris was studied by recombinant protein expression,antibody blocking,RNA interference and Drosophila S2 cell overexpression.These results will help to clear the mechanism of S.eriocheiris infection and host immunity.The study includes the following three aspects: 1.Sequence characteristics,protein expression,antibody preparation and related functions of EsTetraspaninIn this part,the Tetraspanin gene of Eriocheir sinensis was amplified by RACE and named EsTetraspanin.Sequence analysis revealed that EsTetraspanin contains the small extracellular loop(SEL)and a large extracellular loop(LEL)domains and multiple palmitoylation sites.Semi-quantitative PCR and qRT-PCR results showed that EsTetraspanin was the highest level of transcription in hemocytes.The transcription of EsTetraspanin in hemocytes was significantly down-regulated after S.eriocheiris stimulation,indicating that EsTetraspanin plays an important role in the infection of E.sinensis.In addition,the LEL domain of EsTetraspanin was expressed in vitro and polyclonal antibody was prepared,which laid a foundation to further study the immune function of EsTetraspanin.2.Functional study of LEL recombinant protein and polyclonal antibodyThe recombinant protein LEL can be combined with S.eriocheiris in vitro,which lays a foundation to further study the function of the rLEL in S.eriocheiris infection.After co-injection of rLEL with S.eriocheiris,the copy number of S.eriocheiris in hemocytes was significantly reduced,and the mortality of E.sinensis was also significantly dcreased.It indicates that rLEL can attenuate the infective activity of S.eriocheiris.Immunoblotting of hemocytes using polyclonal antibodies revealed that EsTetraspanin was mainly distributed on the surface of hemocytes by laser confocal microscopy.In addition,the S.eriocheiris copy number in the hemocytes was significantly increased after blocking by the EsTetraspanin polyclonal antibody,indicating that the infective activity of S.eriocheiris is significantly improved after antibody blocking.These results indicate that EsTetraspanin plays an important role in the innate immunity of E.sinensis.3.RNA interference of EsTetraspaninIn this experiment,the EsTetraspanin gene was successfully silenced by artificially synthesized dsRNA,and the expression levels of various antimicrobial peptide genes(ALF1,ALF2,ALF3,crustin1)involved in the innate immunity of E.sinensis were significantly down-regulated.After the EsTetraspanin gene was silenced,the S.eriocheiris was used to infect E.sinensis.The results showed that the S.eriocheiris copy number in hemocytes was significantly increased compared with the control group after silencing.At the same time,the mortality of E.sinensis was also significantly increased.This further demonstrates that EsTetraspanin silence could weak the immunity of E.sinensis to S.eriocheiris infection to promote the infection of S.eriocheiris.4.EsTetraspanin-overexpression in Drosophila S2 cellsThe pAc5.1-EsTetraspanin-GFP expression vector was constructed and transferred into Drosophila S2 cells for 24 h.Western blot analysis showed a clear band at 54 kDa.Laser confocal microscope revealed that EsTetraspanin was mainly expressed on the surface of Drosophila S2 cells.After successful overexpression,S.eriocheiris was used to infect Drosophila S2 cells.Compared with the control group,the S.eriocheiris copy number was significantly reduced in the EsTetraspanin overexpression group,and the CCK-8 cell viability assay showed that the EsTetraspanin overexpression group had higher cell viability.In addition,the polyclonal antibodies were used to imprint S.eriocheiris in Drosophila S2 cells.Laser confocal microscopy revealed a significant reduction in the number of S.eriocheiris in Drosophila S2 cells when overexpressed EsTetraspanin.These results indicate that EsTetraspanin increased the host immunity to resist the S.eriocheiris infection.