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Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.

ABSTRACT: BACKGROUND:Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS:The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS:The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P?>?0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P?>?0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS:rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.

SUBMITTER: Hu CX

PROVIDER: S-EPMC7041205 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.

Hu Chen Xi CX Jiang Peng P Yue Xin X Zeng Jie J Zhang Xin Zhuo XZ Song Yan Yan YY Liu Ruo Dan RD Zhang Xi X Wang Zhong Quan ZQ Cui Jing J

Parasites & vectors 20200224 1

<h4>Background</h4>Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis.<h4>Meth ...[more]

PMID: 32093735

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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:Trichinellosis is a serious zoonositc parasitosis worldwide. Because its clinical manifestations aren't specific, the diagnosis of trichinellosis is not easy to be made. Trichinella spiralis muscle larva (ML) excretory-secretory (ES) antigens are the most widely applied diagnostic antigens for human trichinellosis, but the major drawback of the ES antigens for assaying anti-Trichinella antibodies is the false negative in the early Trichinella infection period. The aim of this study was to characterize the T. spiralis putative serine protease (TsSP) and to investigate its potential use for diagnosis of trichinellosis.The full-length TsSP sequence was cloned and expressed, and recombinant TsSP (rTsSP) was purified by Ni-NTA-Sefinose Column. On Western blotting analysis the rTsSP was recognized by T. spiralis-infected mouse serum, and the natural TsSP was identified in T. spiralis ML crude and ES antigens by using anti-rTsSP serum. Expression of TsSP was detected at various T. spiralis developmental stages (newborn larvae, muscle larvae, intestinal infective larvae and adult worms). Immunolocalization identified the TsSP principally in cuticles and stichosomes of the nematode. The sensitivity of rTsSP-ELISA and ES-ELISA was 98.11% (52/53) and 88.68% (47/53) respectively (P > 0.05) when the sera from trichinellosis patients were examined. However, while twenty-one serum samples of trichinellosis patients' sera at 19 days post-infection (dpi) were tested, the sensitivity (95.24%) of rTsSP-ELISA was distinctly higher than 71.43% of ES-ELISA (P < 0.05). The specificity (99.53%) of rTsSP-ELISA was remarkably higher than 91.98% of ES-ELISA (P < 0.01). Only one out of 20 serum samples of cysticercosis patients cross-reacted with the rTsSP. Specific anti-Trichinella IgG in infected mice was first detected by rTsSP-ELISA as soon as 7 dpi and antibody positive rate reached 100% on 10 dpi, whereas the ES-ELISA did not permit detection of 100% of infected mice before 16 dpi.The rTsSP is a potential early diagnostic antigen for human trichinellosis.

Project description:Trichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics.The full-length cDNA encoding a putative cathepsin F-like protease in T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate.Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km?=?0.5091 ?M and Vmax?=?6.12 RFU/s ?M at pH 5.5, and the IC50 value of E64 was 135.50?±?16.90 nM.TsCF1 was expressed in all stages of T. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.

Project description:BackgroundPrevious study showed that Trichinella spiralis proteasome subunit beta type-7 (Tspst) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML), which was screened by using suppression subtractive hybridization (SSH) and confirmed by real-time PCR. Tspst may be related to the larval invasion of intestinal epithelial cells (IECs). The aim of this study was to identify Tspst and to investigate its immune protection against intestinal T. spiralis infection.MethodsThe Tspst gene encoding a 29 kDa protein from T. spiralis infective larvae was cloned, and recombinant Tspst protein (rTspst) was produced in an Escherichia coli expression system. The rTspst was used to immunize BALB/c mice. Anti-rTspst antibodies were used to determine the immunolocolization of Tspst in the parasite. Transcription and expression of Tspst at T. spiralis different developmental stages were observed by RT-PCR and immunofluorescence test (IFT). The in vitro or in vivo immune protection of anti-rTspst serum or rTspst against intestinal T. spiralis infection in BALB/c mice was evaluated.ResultsAnti-rTspst serum recognized the native Tspst protein with 29 kDa in ML crude antigens. Transcription and expression of gene was observed at all T. spiralis different developmental stages (IIL, adult worms, newborn larvae, and ML). An immunolocalization analysis identified Tspst in the cuticle and internal organs of the parasite. An in vitro invasion assay showed that, when anti-rTspst serum, serum of mice infected with T. spiralis or normal mouse serum were added to the medium, the invasion rate of the infective larvae in an IEC monolayer was 25.2%, 11.4%, and 79%, respectively (P < 0.05), indicating that anti-rTspst serum partially prevented the larval invasion of IECs. After a challenge infection with T. spiralis muscle larvae, mice immunized with rTspst conferred a 45.7% reduction in adult worm burden in intestines.ConclusionsIn the present study, Tspst was first identified and characterized. Tspst is an invasion-related protein of T. spiralis IIL and could be considered as a potential vaccine candidate antigen against intestinal T. spiralis infection that merits further study.

Project description:The most commonly used serodiagnostic antigens for trichinellosis are the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML), but the specific antibodies against the ML ES antigens are usually negative during early stage of Trichinella infection. The recent studies demonstrated that T. spiralis adult worm (AW) antigens were recognized by mouse or swine infection sera on Western blot as early as 7-15 days post-infection (dpi), the AW antigens might contain the early diagnostic markers for trichinellosis. The purpose of this study was to screen early diagnostic antigens in T. spiralis AW ES proteins recognized by sera of early patients with trichinellosis. T. spiralis AW were collected at 72 h post-infection (hpi), and their ES antigens were analyzed by SDS-PAGE and Western blot. Our results showed that 5 protein bands (55, 48-50, 45, 44, and 36 kDa) were recognized by sera of early patients with trichinellosis collected at 19 dpi, and were subjected to shotgun LC-MS/MS and bioinformatics analyses. A total of 185 proteins were identified from T. spiralis protein database, of which 116 (67.2%) proteins had molecular weights of 30?60 kDa, and 125 (67.6%) proteins with pI 4-7. Bioinformatic analyses showed that the identified proteins have a wide diversity of biological functions (binding of nucleotides, proteins, ions, carbohydrates, and lipids; hydrolase, transferase, and oxidoreductase, etc.). Several enzymes (e.g., adult-specific DNase II, serine protease and serine protease inhibitor) could be the invasion-related proteins and early diagnostic markers for trichinellosis. Moreover, recombinant T. spiralis serine protease (rTsSP-ZH68) was expressed in E. coli and its antigenicity was analyzed by Western blot with the early infection sera. The rTsSP-ZH68 was recognized by sera of infected mice at 8-10 dpi and sera of early patients with trichinellosis at 19 dpi. T. spiralis AW proteins identified in this study, especially serine protease, are the promising early diagnostic antigens and vaccine candidates for trichinellosis.

Dataset Information

Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.

Publications

Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.

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