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Serodiagnostic antigens of Clonorchis sinensis identified and evaluated by high-throughput proteogenomics.

ABSTRACT: Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis.

SUBMITTER: Cho PY

PROVIDER: S-EPMC7793300 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Serodiagnostic antigens of Clonorchis sinensis identified and evaluated by high-throughput proteogenomics.

Cho Pyo Yun PY Lee Ji-Yun JY Kim Tae Im TI Song Jin-Ho JH Hong Sung-Jong SJ Yoo Won Gi WG Tsuboi Takafumi T Ha Kwon-Soo KS Jung Jae-Wan JW Takeo Satoru S Han Eun-Taek ET Sripa Banchob B Hong Sung-Tae ST Chai Jong-Yil JY Nam Ho-Woo HW Pak Jhang Ho JH Kim Tong-Soo TS

PLoS neglected tropical diseases 20201228 12

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected an ...[more]

PMID: 33370333

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Project description:BackgroundClonorchiasis, caused by the liver fluke Clonorchis sinensis, remains a serious public health issue in Asia, especially in China, and its relationship with cholangiocarcinoma has highlighted the importance of C. sinensis infection. Proteins containing tandem repeats (TRs) are found in a variety of parasites and, as targets of B-cell responses, are valuable for the serodiagnosis of parasite infections. Here, we identified a novel C. sinensis-specific antigen, Cs1, containing TRs, and investigated its diagnostic value, other immunological properties, and tissue distribution.Methodology/principal findingsA partial Cs1 cDNA sequence was cloned by screening an adult C. sinensis cDNA expression library. The full-length Cs1 cDNA was obtained by 5' rapid amplification of cDNA ends. The deduced Cs1 protein consists of a signal peptide and five TRs of 21 amino acids. The recombinant Cs1 (rCs1) was constructed and purified. rCs1 showed higher sensitivity (94.3%) and specificity (94.4%) than the C. sinensis excretory-secretory products (ESPs) according to ELISA of 114 serum samples. Native Cs1 was identified in C. sinensis ESPs and crude antigens of adult C. sinensis by western blotting using an anti-rCs1 monoclonal antibody. ELISA of recombinant peptides of different Cs1 regions demonstrated that the TR region was immunodominant in Cs1. Immunohistochemistry and confocal microscopy revealed that Cs1 is located in a granule-like structure surrounding the acetabulum of C. sinensis adults that has not previously been described.Conclusions/significanceWe identified a novel C. sinensis-specific TR protein, Cs1, which is an antigen of high serological significance, compared with C. sinensis ESPs. The deduced features of Cs1 show a unique structure containing TRs and a signal peptide and the TR region is immunodominant in Cs1. This provides a basis for targeted screens of other antigens. The novel structure in which Cs1 is located also deserves further investigation.

Project description:High-intensity clonorchiasis infection is associated with serious outcomes, including cancer. Understanding the infection intensity of Clonorchis sinensis and its risk factors in local endemic regions could facilitate effective control measures. In a county located in a highly endemic area in Guangxi Province, P. R. China, local residents were randomly enrolled in the study; helminth egg examinations were performed with the Kato-Katz method, and the intensity of infection was identified as mild, moderate or heavy. Knowledge, attitudes, and high-risk behaviours were investigated among those infected with Clonorchis sinensis. A total of 2521 local residents participated in this study, and the Clonorchis sinensis-positive proportion was 28.9% (728 persons). Among the infected persons, the percentages of mild, moderate and heavy infections were 66.2%, 28.4% and 5.4%, respectively. Males experienced a higher proportion of moderate and heavy infections (37.5%) than females (18.1%) (p<0.05). The highest infection proportion among the different levels of infection intensity was identified among persons aged 30-59 years (15.7% for moderate and heavy infections). Among the 509 persons who reported eating raw fish, 302 persons (59.3%) had eaten raw fresh fish for more than 10 years, and 131 (25.7%) persons ate raw fish ?12 times a year. Multivariate logistic regression revealed that eating raw fish 12-50 times in the last year (adjusted odds ratio [aOR] = 1.74, 95%CI: 1.09-2.80) and eating raw fish >50 times in the last year (aOR = 2.89, 95%CI: 1.20-7.50) were risk factors for high-intensity infections (moderate and heavy). The overall infection proportion was high in the study area, with a large group of residents experiencing high-intensity infections. High frequency of raw fish consumption was associated with high-intensity infections. Intervention strategies targeting people with a high frequency of raw fish consumption should be implemented to reduce the probability of severe consequences.

Project description:Clonorchiasis, which is caused by Clonorchis sinensis, is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of C. sinensis play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for C. sinensis were collected to identify proteins specific to the sera of C. sinensis (i.e., proteins that do not cross-react with Fasciola hepatica and Schistosoma japonicum) at different infection periods. Briefly, white Japanese rabbits were artificially infected with C. sinensis, and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in C. sinensis, a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of C. sinensis, F. hepatica, and S. japonicum. For the annotated proteins, 32, 18, 39, and 35 proteins specific to C. sinensis were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with C. sinensis. No cross-reaction occurred with the positive sera of S. japonica, F. hepatica, and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis.

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Serodiagnostic antigens of Clonorchis sinensis identified and evaluated by high-throughput proteogenomics.

Publications

Serodiagnostic antigens of Clonorchis sinensis identified and evaluated by high-throughput proteogenomics.

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