| The abuse of antibiotics leads to the pollution of antibiotics in the environment,and also aggravates the generation of bacterial resistance.It is reported that the existing antibiotics have different degrees of resistance,so it is urgent to develop new antibacterial drugs to deal with the problem of bacterial resistance.Antimicrobial peptides are a kind of important immune factors in the innate immune system and play an important role in resistance to infection of pathogenic microorganism.It has broad spectrum antimicrobial activity and multiple mechanisms,and is not easy to cause drug resistance of pathogenic bacteria.Therefore,they are considered as an ideal substitute for antibiotics and can be developed as new antibacterial drugs.At present,most of the known antimicrobial peptides come from terrestrial organisms,and the number of antimicrobial peptides derived from marine animals is very few.The mud crab,Scylla paramamosain,is an important mariculture species in southern China,which has important economic value.Mud crab has an open system,and multiple molting processes before mature.It exposed in sea water and vulnerable to the infection of marine microorganisms.To maintain healthy development,there may be more innate immune factors in mud crab and antimicrobial peptide is important congenital immune factors,thus mud crab may be an important source for isolation and identification of novel antimicrobial peptides.In this study,we screened for genes related to immune response from the macroeye juvenile transcriptome of mud crab,and three genes were found to be unannotated new genes,named Sparanegtin,Spamprin and Spamplin respectively.By predicting its antibacterial domain,the peptides with antimicrobial activity were screened.In this paper,the expression characteristics of the three genes,the antimicrobial activities and mechanisms of recombinant proteins and synthetic peptides and their immune-related functions were studied in depth.The results are as follows:1.The coding sequence and physicochemical properties of three genes were elucidated.The GenBank accession number of Sparanegtin gene was MN612064,and the cDNA was 525 bp,the 5’ non-coding region was 44 bp,the Open reading frame was 252 bp,and the 3’ non-coding region was 229 bp.The signal peptide contains 23 amino acids,and the mature peptide contains 60 amino acids.The theoretical isoelectric point of mature peptide is 5.21,which is a kind of anionic polypeptide.The GenBank accession number of Spamprin gene was MT707606,with a cDNA of 589 bp,a 5’ noncoding region of 102 bp,an ORF frame of 291 bp,and a 3’ non-coding region of 196 bp.It encodes 96 amino acids and has no signal peptide structure.The theoretical isoelectric point of mature peptide is 10.15.It is a cationic polypeptide.The GenBank accession number of Spamplin gene was MW388711,with a cDNA of 575 bp,a 5’ noncoding region of 18 bp,an ORF frame of 345 bp,and a 3’ non-coding region of 212 bp.It encodes 114 amino acids and has no signal peptide structure.The theoretical isoelectric point of mature peptide is 8.93.It is a cationic polypeptide.2.The expression characteristics of three genes in normal tissues of male and female crabs,different developmental stages and molting stage of juvenile crabs were revealed.Sparanegtin gene was only highly expressed in testicle of male crab,and the expression levels were low in other tissues of male and female crab.Spamprin gene was highly expressed in both male and female crabs,mainly in the testis,gills and eye stalks of male crabs and in the nerve masses,stomach and brain of female crabs.Spamplin gene is highly expressed in the posterior ejaculatory ducts and testis of male crabs,and also in the gills,stomach,heart and ovaries of female crabs.In the development stage of blue crab,the expression patterns of the three genes all reached the maximum expression in the young crab stage,but there were differences in the expression of flea-like larva and big-eye larva.Sparanegtin gene was widely expressed in zoeal larvae from stage Ⅳ.Spamprin gene was expressed in large numbers from larva stage.The expression of Spamplin gene was high in zoeal Ⅴ stage,but low in macroeye stage.Sparanegtin gene was significantly up-regulated after molting at stageⅠ,Ⅱ and Ⅲ.Spamplin gene was significantly upregulated after molting of juvenile crabs at stage Ⅰ and stage Ⅲ,indicating that it played an important role in the molting process of crabs.3.The expression characteristics of three genes in different tissues and organs after LPS stimulation and Vibrio alginolyticus infection were revealed.In testis,Sparanegtin gene was significantly down-regulated after LPS stimulation,while Spamprin and Spamplin genes were significantly up-regulated.Spamprin gene was significantly down-regulated after V.alginolyticus infection,while Sparanegtin gene and Spamplin gene were significantly up-regulated.Sparanegtin gene was significantly up-regulated in blood cells after LPS stimulation and V alginolyticus infection.Both Spamprin and Spamplin genes were significantly up-regulated in gill tissue after LPS stimulation.The expression of Spamprin did not change after V alginolyticus infection,but the expression of Spamplin was significantly up-regulated.Sparanegtin,Spamprin and Spamplin genes all responded to LPS stimulation and V alginolyticus infection in the tested tissues,and each gene had different expression patterns.4.The prokaryotic expression vector of Sparanegtin gene was constructed and its expression product had strong antimicrobial activity.Sparanegtin mature peptide recombinant protein was expressed and purified by constructing the prokaryotic expression vector of Sparanegtin mature peptide.And the minimum inhibitory concentration of rSparanegtin against Pseudomonas aeruginosa,Pseudomonas fluorescens,Aeromonas hydrophila and Shigella flexneri and Staphylococcus aureus were of 12-24 μM;the minimum inhibitory concentration of rSparanegtin against Escherichia coli,Staphylococcus epidermidis,Cryptococcus neoformans and Pichia were 24-48 μM.rSparanegtin could effectively inhibit spore germination of Aspergillus fumigatus,Aspergillus niger and Aspergillus ochraceus at a certain concentration.When the concentration was 48 μM,rSparanegtin could completely kill E.coli at 5 h,and P.aeruginosa at 4 h.rSparanegtin had strong concentration dependent binding ability to Lipopolyaccharides,Peptidoglycan and Lipoteichoic acid.The binding ability of LPS was higher than that of PGN and LTA.rSparanegtin has strong binding ability to P.aeruginosa and E.coli.The cell membrane integrity of P.aeruginosa and A.ochratos spores was obviously damaged by rSparanegtin treatment,resulting in cell content leakage.5.It was revealed that Sparanegtin expression product had immune protection effect in vivo.The results showed that rSparanegtin had no obvious cytotoxicity.rSparanegtin treatment can significantly reduce the endotoxin level of V alginolyticus in a dose-dependent manner.rSparanegtin did not show activity against V.alginolyticus in vitro.After pre-incubation with rSparanegtin and V.alginolyticus,S.paramamosain were injected.After 5 days of observation,40%of S.paramamosain survived in the rSparanegtin treatment group,while all of them died in the control group,indicating that rSparanegtin can significantly improve the survival rate of S.paramamosain.It was also found that the number of bacteria in midgut,gills and hepatopancreas treated with rSparanegtin was significantly reduced.Transcriptional changes of several immunerelated genes were induced,such as immune-related genes(SpToll2,SpMyd88,SpSpaetzle and SpSTAT),AMPs(SpHyastatin and SpALF2)and antioxidant related genes(SpSOD,SpCAT and SpGPx).6.Two antimicrobial peptides Spamprin4-23 and Spamplin58-82 were screened to reveal their antimicrobial activity and mechanism.Based on the amino acid sequences of Spamprin and Spamplin,the antimicrobial functional domain was analyzed by bioinformatics,and the truncated peptides Spamprin4-23 and Spamplin58-82 were obtained.The results showed that the minimum bactericidal concentration of Spamprin4-23 against P.stutzeri,S.flexneri and Pichia were 3-6 μM.The minimum bactericidal concentration of B.subtilis,F.oxysporum and F.solani were 12-24 μM.The minimum bactericidal concentrations of S.aureus,E.coli,P.aeruginosa,C.neoformococcus,A.niger and A.ochraceus were 48-96 μM.The minimum bactericidal concentration of Spamplin58-82 against Enterococcus faecium was 1.5-3 μM.The minimum bactericidal concentration of Staphylococcus epidermidis,Listeria monocytogenes and S.flexneri were 3-6 μM.The minimum bactericidal concentrations of S.aureus,A.baumannii,C.neofordii,Pichia,F.oxysporum and F.solani were 1224μM.The minimum bactericidal concentrations of Bacillus cereus,Pseudomonas schisterii,Fusarium oxysporum,Aspergillus Niger and Aspergillus ochratus were 2448μM.The minimum bactericidal concentration of Escherichia coli and Pseudomonas aeruginosa was 48-96 μM.The results showed that both Spamprin4-23 and Spamplin5882 had broad spectrum antimicrobial activities,but there were differences in antimicrobial activities.The antimicrobial types and inhibitory concentration of Spamprin4-23 and Spamplin58-82 had different antimicrobial activities against the same bacteria.Both Spamprin4-23 and Spamplin58-82 could effectively inhibit A.fumigatus,A.ochratus,A.niger,F.graminearum,F.oxysporum and F.oxysporum.Spamprin4-23 and Spamplin58-82 showed different bactericidal rates:when Spamprin4-23 concentration was 6 μM,P.stutzeri could be completely killed at 30 min.At the concentration of Spamprin4-23 of 6 and 12 μM,S.flexneri could be completely killed at 4h.When Spamplin58-82 concentration was 3 and 6 μM,E.faecium could be completely killed at 360 min.When Spamplin58-82 concentration was 12 μM,L.monocytogenes could be completely killed at 90 min.When the concentration of Spamplin58-82 was 24μM,acinetobacter baumannii was completely killed at 60 min.Spamprin4-23 can increase the production of Reactive oxygen species in S.flexneri and P.stutzeri in a concentration dependent manner.It can also inhibit CAT activity in bacteria.Spamprin4-23 binds to the DNA of bacteria;Confocal action site analysis showed that Spamprin4-23 and Spamplin58-82 acted on the entire bacteria,not only acting on the surface of the bacteria,but also entering the bacteria.After bacteria incubated with Spamprin4-23,cell permeability changed and intracellular substances leaked out.The results of scanning electron microscopy showed that the spores of Spamprin4-23 coincubated bacteria and fungi showed cell shrinkage and the contents of broken cells were released.After Spamplin58-82 co-incubated,all the bacteria showed the phenomenon of bacterial aggregation,shrinkage and loss of cell morphology,and the contents of cell fragmentation were released.7.It was revealed that Spamprin4-23 had immune protective effect in vivo.It was found that Spamprin4-23 had no cytotoxic.Spampriin4-23 did not show activity against V.alginolyticus in vitro.After pre-incubation with Spamprin4-23 and V alginolyticus,S.paramamosain were injected.It was found that Spamprin4-23 group could reduce the death rate of crabs,and 30%of the Spamprin4-23 group still survived when all the control group died.It was also found that the number of bacteria in midgut,gills and hepatopancreas of crabs treated with Spamprin4-23 was significantly reduced.We found that the expression levels of immune-related genes(SpRelish and SpSTAT),antioxidation-related genes(SpCAT and SpGPx),antimicrobial peptides(SpALF6)and cytokines(SpLITAF)increased significantly.8.The immune signaling pathway factors related to Spamplin gene expression were analyzed.The results showed that all crabs in the dsSpamplin group died 96 h after infection with V.alginolyticus,while 13%of crabs in the dsGFP group survived 120 h after infection,suggesting that knockdown with Spamplin gene can reduce the survival rate of V.alginolyticus infection in S.paramamosain.Spamplin gene can regulate the expression levels of immune-related genes SpSpaetzle,SpTolll,SpToll3,SpToll6,SpCrustinl and SpCrutin5.After in vivo knockdown with Spamplin,the expression levels of Toll pathway genes SpToll1,SpToll3 and SpToll6 were significantly up-regulated,and the expression levels of antimicrobial peptides SpCrustinl and SpCrutin5 were significantly up-regulated in blood cells.The expression of Toll pathway gene SpTolll and antimicrobial peptide gene SpCrustin1 were significantly up-regulated in midgut tissue.In muscle tissue,the expression of Toll pathway gene SpToll1 and antimicrobial peptide gene SpCrustin5 were significantly upregulated.After the expression of Spamplin gene was interfered,the regulation of immune gene expression was different in different tissues.After the expression of Spamplin gene is suppressed,there may be a compensation mechanism by upregulating the expression of SpToll gene and regulating the expression of the downstream antimicrobial peptide SpCrustin to compensate the immune response.This study laid a foundation for further development of the immune protection mechanism of Spamplin in vivo. |
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