A Functional Study Of Actin-like MreB In The Morphological Changes And Pathogenesis Of Spiroplasma Sinensis

Spiroplasma eriocheiris,a novel aquatic crustacean pathogenic microorganism,is also the first spiroplasma to be discovered in the aquatic environment.It has been proved to be the pathogen of tremor disease in Chinese mitten crab Eriocheir sinensis.In addition to E.sinensis,it has also been proved to be harmful to Macrobrachium rosenbergii,Macrobrachium nipponensis,Procambarus clarkii and other aquatic crustaceans,threatening the sustainable development of aquaculture.S.eriocheiris is a kind of bacterium with unique movement and spiral shape in Mollicutes.It has no cell wall,its spiral shape mainly depends on the inside cytoskeleton.This study mainly used recombination protein expression and purification,Western blot,real-time quantitative fluorescent PCR,flow cytometry and other technologies to treat the S.eriocheiris with the agonists(phalloidin)and inhibitor(A22)of the spiroplasma skeletal protein Mre B,to explore the functions of Mre B in the morphology and pathogenicity of spiroplasma.The results provided new targets and more effective prevention and control methods for blocking the pathogen.This study mainly includes the following three results:1.Expression,antibody preparation and detection of Mre B of S.eriocheirisThe pET-30a plasmids containing the Mre B1-5 genes previously constructed by our group were transformed into E.coli for recombination protein expression.Except for Mre B3,4 types of Mre B,namely Mre B1,Mre B2,Mre B4 and Mre B5 were successfully obtained.The molecular weight of Mre B1 and Mre B2 were both 37.9k Da,Mre B4 was 40.7k Da,and Mre B5 was 38.5k Da.Rabbit polyclonal antibodies were prepared after purification of the 4 successfully expressed Mre B recombinant proteins.Since the specificity of the antibodies was not as well as expected,specific peptides of Mre B1-5 were chemical synthesis to prepare mouse monoclonal antibodies.Among them,the Mre B3,Mre B4,and Mre B5 antibodies were good specificity and meet the needs of subsequent experiments.2.Roles of Mre B in morphology and cytoskeleton of S.eriocheirisS.eriocheiris presents a typical spiral shape in PBS,but was spherical in water.After treated with 0.1%Triton,the skeleton of S.eriocheiris was consistent with its morphology,that was,the spiral shape of S.eriocheiris was a long spiral shape,while the spherical skeleton of it was round,which was consistent with the spherical shape.The results of immunoelectron microscopy showed that Mre B3,Mre B4,and Mre B5are distributed on the outer surface of the cell membrane of S.eriocheiris.Western blot results showed that there was no significant difference in the expression of Mre B5 when S.eriocheiris was in spiral and spherical shape,while the expression of Mre B3 and Mre B4 in spiral shape were significantly higher than that in spherical shape.After treated with 2.5?g/ml phalloidin and 200?g/ml A22 respectively,the results with electron transmission microscopy showed that the length of S.eriocheiris in the phalloidin treatment was significantly increased,while the length of the A22 treatment group was significantly shortened and approximately spherical.And the expressions of Mre B3,Mre B4,and Mre B5 in the phalloidin treatment group were not changed significantly,the Mre B3 and Mre B5 in the A22 treatment group were not significant changed,too,but the expression of Mre B4 was significantly lower than that of control.3.Study on the pathogenicity of S.eriocheiris treated with phalloidin and A22The CCU results proved that phalloidin had no effect on the growth rate and copy number of S.eriocheiris.Whereas A22 could significantly delay the growth rate of it,delaying the logarithmic phase that could be achieved in 12 h to 8 days,but the copy number of S.eriocheiris had no significant change at the logarithmic phase and can also reach 10~8 like the control.Compared with control group,there was no significant difference after S.eriocheiris treated with phalloidin.But 49.5%of the S.eriocheiris treated with A22 dead,and would reduce the mortality of crabs.After phalloidin-treated S.eriocheiris infected,the morphology of hemocytes was destroyed,cell viability was significantly reduced,phenoloxidase(PO)activity was significantly increased,and the S.eriocheiris copy number and cell apoptosis significantly increased.Compared with the control,after the A22-treated S.eriocheiris infecting hemocytes,although some hemocytes were destroyed,but some were still normal,and the cell viability was significantly increased.Besides,the copy number of S.eriocheiris was significantly reduced,PO activity was significantly increased,and the number of cell apoptosis decreased significantly.The above results showed that under the treat of the promoter and inhibitor of the cytoskeleton Mre B,the pathogenicity of S.eriocheiris changed significantly.The pathogenicity of S.eriocheiris was enhanced significantly after the phalloidin treatment,while reduced significantly after treated with inhibitor A22.