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Characterization of Two Trichinella spiralis Adult-Specific DNase II and Their Capacity to Induce Protective Immunity.

ABSTRACT: Deoxyribonuclease II (DNase II) is a widespread endonuclease, which can degrade the DNA. Trichinella spiralis adult-specific DNase II-1 (TsDNase II-1) and DNase II-7 (TsDNase II-7) were identified in excretory-secretory (ES) or surface proteins of adult worm (AW) and intestinal infective larvae (IIL) using immunoproteomics with early infection sera. The aim of this study was to characterize the two T. spiralis DNase II enzymes and to investigate their role as potential vaccine candidate target molecules. The cDNA sequences of the two DNase II enzymes from 3 days old AWs of T. spiralis were cloned and expressed. The sequencing results showed that the complete cDNA sequences of the two DNase II enzymes were 1221 and 1161 bp long, and the predicted open reading frames encoded 347 and 348 amino acids, respectively. On Western blot analysis, natural TsDNase II-1 and TsDNase II-7 in the crude extracts of IIL, AWs, and newborn larvae (NBL) and AW ES proteins were recognized by both anti-rTsDNase II-1 and anti-rTsDNase II-7 sera. Indirect immunofluorescence test and qPCR showed that the two DNase II enzymes were highly expressed at AW and NBL stages and were mainly located at the cuticle and stichosome of the nematode. Vaccination with the two recombinant DNase II enzymes triggered prominent humoral responses that exhibited significant immune protection against T. spiralis larval infection, as demonstrated by the notable reduction in intestinal AW and muscle larva burdens. Specific antibodies to the two molecules evidently inhibited the in vitro parasite invasion of enterocytes and participated in the killing of NBL by an antibody-dependent cell-mediated cytotoxicity (ADCC) mode. The enzymes DNase II-1 and DNase II-7 are the potential target molecules for anti-Trichinella vaccine for blocking both larval invasion and development.

SUBMITTER: Qi X

PROVIDER: S-EPMC6230719 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Characterization of Two Trichinella spiralis Adult-Specific DNase II and Their Capacity to Induce Protective Immunity.

Qi Xin X Yue Xin X Han Yue Y Jiang Peng P Yang Fan F Lei Jun J JJ Liu Ruo D RD Zhang Xi X Wang Zhong Q ZQ Cui Jing J

Frontiers in microbiology 20181105

Deoxyribonuclease II (DNase II) is a widespread endonuclease, which can degrade the DNA. Trichinella spiralis adult-specific DNase II-1 (TsDNase II-1) and DNase II-7 (TsDNase II-7) were identified in excretory-secretory (ES) or surface proteins of adult worm (AW) and intestinal infective larvae (IIL) using immunoproteomics with early infection sera. The aim of this study was to characterize the two T. spiralis DNase II enzymes and to investigate their role as potential vaccine cand ...[more]

PMID: 30455671

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Project description:BackgroundDeoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.Methodology/principal findingsIn this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.ConclusionsThis study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.

Project description:BackgroundNudix hydrolases (Nd) is a widespread superfamily, which is found in all classes of organism, hydrolyse a wide range of organic pyrophosphates and has a 'housecleaning' function. The previous study showed that Trichinella spiralis Nd (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with T7 phage-displayed TsNd polypeptides produced protective immunity. The aim of this study was to clone, express and identify the full-length TsNd and to investigate its immune protection against T. spiralis infection.MethodsThe full-length cDNA sequence of TsNd gene encoding a 46 kDa protein from T. spiralis intestinal infective larvae (IIL) was cloned and identified. The antigenicity of rTsNd was analyzed by Western blot. Transcription and expression of TsNd at T. spiralis different stages were observed by RT-PCR and IFT. The levels of the specific total IgG, IgG1 and IgG2a antibodies to rTsNd were determined by ELISA. The immune protection of rTsNd against T. spiralis infection was investigated.ResultsSequence and phylogenetic analysis revealed that TsNd had a nudix motif located at 226-244aa, which had high homology and the closest evolutionary status with T. pseudospiralis. The rTsNd was obtained after expression and purification. Western blot analysis showed that anti-rTsNd serum recognized the native TsNd protein in crude antigens of muscle larvae (ML), IIL, adult worms (AW) and newborn larvae (NBL), and ES antigens of ML. Transcription and expression of TsNd gene was observed in all developmental stages of T. spiralis (ML, IIL, AW and NBL), with high level expression in IIL. An immunolocalization analysis identified TsNd in the cuticle, stichocytes and reproductive organs of the parasite. Following immunization, anti-rTsNd IgG levels were increased, and the levels of IgG1 were more significantly higher than that of IgG2a. After a challenge infection with T. spiralis, mice immunized with the rTsNd displayed a 57.7% reduction in adult worms and a 56.9% reduction in muscle larval burden.ConclusionsTsNd induced a partial protective immunity in mice and could be considered as a novel candidate vaccine antigen against trichinellosis.

Project description:Glutathione-S-transferase (GST) is a widespread multigene family of detoxification enzymes. The vaccination of mice with recombinant GST of 24 kDa from Trichinella spiralis elicited a low immune protection against challenge infection. The objective of this study was to characterize the T. spiralis putative GST gene (TspGST) encoding a 30.8 kDa protein and to evaluate its potential as a candidate antigen for anti-Trichinella vaccine.The full-length cDNA sequence of TspGST from T. spiralis muscle larvae (ML) was expressed in E. coli. The enzymatic activity and antigenicity of the rTspGST were identified by spectrophotometry, Western blot, and ELISA. The expression of TspGST at T. spiralis various stages was investigated by RT-PCR and indirect immunofluorescent test (IIFT). Serum level of total IgG, IgG1, and IgG2a antibodies against rTspGST were measured by ELISA. The immune protection produced by vaccination with rTspGST against T. spiralis was evaluated.The sequencing results showed that the cDNA of TspGST was 840 bp, and encoded a protein of 279 amino acids, which had a molecular size of 30.8 kDa and a pI of 5.21. Its amino acid sequence shares 37% similarity with TsGST. The rTspGST protein had enzymatic activity of GST. On Western blot and ELISA analysis, the native TspGST protein with 30.8 kDa in crude antigens derived from adult worms (AW), newborn larvae (NBL), infective intestinal larvae (IIL) and ML was recognized by anti-rTspGST sera, but the ML ES antigens could be not recognized by anti-rTspGST sera. Expression of TspGST was found in all of T. spiralis various stages (AW, NBL, ML, and IIL). An immunolocalization analysis identified TspGST in different stages (mainly in cuticles) of the nematode. The mice vaccinated with the rTspGST elicited Th2-predominant immune responses, showed a 34.38% reduction of adult worms and a 43.70% reduction of muscle larvae.Immunization with rTspGST produced a partial immune protection, and the rTspGST could be regarded as a potential candidate target for an anti-Trichinella vaccine.

Dataset Information

Characterization of Two Trichinella spiralis Adult-Specific DNase II and Their Capacity to Induce Protective Immunity.

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Characterization of Two <i>Trichinella spiralis</i> Adult-Specific DNase II and Their Capacity to Induce Protective Immunity.

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