| Background and PurposeTrichinellosis is a serious food-borne zoonotic parasitic disease with global distribution,causing serious harm to public health and food safety.Trichinella spiralis undergoes four molts for growth and development.It is of great significance to study the mechanism of T.spiralis molting related proteins for screening anti-Trichinella drugs and developing vaccines.In this study,mass spectrometry was used to identify hydrolytic proteases in the excretory secretion(ES)proteins of T.spiralis,the proteomic differences between muscle larvae(ML)and intestinal infective larva(ⅡL)in molting stage were compared.Bioinformatics analysis was conducted to screen out the molting related proteins.These proteins may be cuticle structural proteins or involved in the remodeling and degradation of cuticle.Metalloproteinase plays an important role in the molting process of T.spiralis.In this study,the metalloproteinase Tsdpy31 identified by mass spectrometry was cloned,expressed and purified.Real-time PCR and Western blot were used to detect the levels of Tsdpy31 gene transcription and protein expression,and immunofluorescence assay was used to identify its localization in the worm.The enzyme activity of rTsdpy31 and its mutants were studied by specific substrate.The function of Tsdpy31 in the molting process of T.spiralis was identified using RNAi technique.The results of this study are helpful to elucidate the biological characteristics of Tsdpy31 which plays an important role in the molting process of T.spiralis,and may lay a foundation for the development of new anti-intestinal T.spiralis vaccines or drugs.Materials and Methods1.Trichinella strain and experimental animalsTrichinella spiralis strain(ISS534)was kept by mice serial passaging in our department.Specific-pathogen-free(SPF)6-week old mice were obtained from the Henan provincial laboratory animal center.2.Screening of molting-related proteins from Trichinella spiralis ES protein andsoluble protein.The ES protein of Trichinella spiralis ML and ⅡL was collected.The enzyme activity of ES protein was determined by azocasein substrate and gelatin zymography.And after the ES protein was incubated with different kinds of protease inhibitors,the enzyme activity was detected.Protein bands that could hydrolyze gelatin were analyzed by mass spectrometry to identify the proteolytic enzymes.These proteins were analyzed by bioinformatics to screen out the proteases that might be related to molt.At the same time,muscle larvae and 10 h ⅡL larvae in the first molting stage were collected and the crude proteins were prepared for quantitative proteomic analysis.Real-time PCR was used to analyze the transcriptional levels of ES proteases and molting-related proteins in 10 h ⅡL.3.Biological and functional characterization of the metalloproteinases Tsdpy31 from Trichinella spiralis.The molting-related protein metalloproteinase Tsdpy31 which was identified by mass spectrometry was selected to study.Specific primers were designed to amplify Tsdpy31 gene using ⅡL cDNA as template,and rTsdpy31 was obtained by prokaryotic expression.The Tsdpy31 gene transcription,protein expression level and the localization in Trichinella were analyzed by Real-time PCR,Western blot and immunofluorescence assay.The enzyme activity of rTsdpy31 was detected using specificity substrate.The active sites of Tsdpy31 were mutated,and then,the mutant activity was detected.In addition,the T.spiralis collagen Tscol2 was expressed and purified which specifically expressed in T.spiralis 10 h ⅡL.The specific binding between Tscol2 and Tsdpy31 was detected by Far Western blot and ELISA.4.Effects of Tsdpy31 gene silenced by RNAi on molting and development of worms.The mice were infected orally with T.spiralis muscle larvae after Tsdpy31 gene was silenced by RNAi.The morphology of ⅡL was observed after first molting,and the cuticle permeability of worms in different groups was detected by nuclear fluorescent dye.The molting sheath of the larvae was observed and counted to determine the function of Tsdpy31 in the molting of T.spiralis.Intestinal 3d and 6d adult worm burden and 35d muscle larval worm burden after infection were detected.Females from each group were cultured,and the reproductive capacity of female AW was ascertained in line with the NBL produced by each female in 72 h.The lengths of worms at different stages were measured.And the differences in body lengths were analyzed.5.Statistical analysisAll data was analyzed with SPSS 20.0 software.One-way ANOVA,Chi square test and linear regression were mainly used to analyze the data.P<0.05 was defined as statistical significance.Results1.The molting-related proteins in Trichinella spiralis ES and the crude protein.Thirty proteinases were identified by mass spectrometry from the Trichinella spiralis ⅡL ES.There are 19 serine proteinases,7 metalloproteinases and 3 cysteine proteinases.Extensive proteomic information in Trichinella molting larvae was established.Through bioinformatics analysis,323 differential proteins were screened by LC-MS/MS.These proteins may be involved in the synthesis,maintenance,remodeling and degradation of the cuticle,or in hormone regulation related to molting and intracellular transportation and regulation.2.Biological and functional characterization of Trichinella spiralis metalloproteinase Tsdpy31.Tsdpy31 was transcribed and expressed at different life-cycle stages of Trichinella spiralis and located principally at the cuticle,stichosome,hypodermis and embryo of the parasite.The transcriptional level was high at 6 h ⅡL in proecdysis,15 h ⅡL in the second molting stage and 6 d adults.The enzymatic activity analysis showed that rTsdpy31 had the catalytic activity of natural metalloproteinase.The optimal reaction pH and temperature were 8.0 and 37 ℃.The enzymatic activity of rTsdpy31 was significantly enhanced by Zn2+ metal ions.The inhibitor EDTA has an inhibitory effect on rTsdpy31 enzyme activity.After mutating the active site of Tsdpy31,the enzyme activity of MTsdpy31 was completely lost.Far-Western and ELISA results indicated that there is a specific binding between Tsdpy31 and Tscol2 proteins.3.The silencing of Tsdpy31 by RNAi enhances the permeability of new cuticle and reduces the fecundity of female worms.Mice were inoculated with T.spiralis larvae transfected with Tsdpy31 dsRNA.15 h ⅡL were collected after the first molting.It was found that a nuclear fluorescent dye could penetrate the larvae cuticle and dye them.The results showed that RNA interference enhanced the permeability of new cuticle.RNAi displayed a 34.72%reduction in 6 d adult worms and 53.21%reduction in ML burden.The length of 3 d female larvae and newborn larvae decreased 10.07 and 4.55%(P<0.05).Compared with PBS group,female fecundity decreased 61.71%(P<0.05).Conclusions1.The molting-related proteins were screened by mass spectrometry and bioinformatics analysis from Trichinella spiralis ES proteins and crude proteins.2.Tsdpy31 was transcribed and expressed at different life-cycle stages of T.spiralis and mainly located at the cuticle,stichosome,hypodermis and embryo.The transcriptional levels were increased at before the larval molting period and 6 d adults.Tsdpy31 has the hydrolytic activity which natural metalloproteinases possess.Tsdpy31 can specifically bind to the T.spiralis cuticle collagen protein rTscol2.3.Tsdpy31 is involved in the formation of new cuticles and the peeling of old cuticles during Trichinella molting,and affects the development and reproduction of Trichinella spiralis. |
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