Identification of Myoferlin, a Potential Serodiagnostic Antigen of Clonorchiasis, via Immunoproteomic Analysis of Sera From Different Infection Periods and Excretory-Secretory Products of Clonorchis sinensis

Abstract

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.

Clonorchis sinensis infection modulates key cytokines for essential immune response impacted by sex

“…C. sinensis infection is carcinogenic to human [26][27][28]. The pathogenic mechanisms include mechanical injury of biliary epithelia by the flukes, immunopathological changes caused by infectionrelated inflammation, and direct effects of the excretory-secretory products (ESPs) [8,[29][30][31]. Here we propose, besides above 3 mechanisms, C. sinensis infection caused immune suppression might also facilitate the transformation and proliferation of the tumor cells.…”

A point-of-care testing assay for clonorchiasis using a EuNPs-CsTR1 fluorescent probe-based immunoassay

“…Adult worms were collected from the bile ducts of infected rabbits 5 weeks post-infection. Excretory-secretory products (ESPs) and crude antigens of adult worms were prepared as previously described [ 28 ]. A total of 151 human sera were used in this study, 59 serum samples were collected from patients with clonorchiasis (egg-positive as detected by the KK and PCR method), and healthy individuals without parasitic infections (n = 74), 9 mixed infection sera (2 cases of mixed infection sera of C .…”

“…Adult worms were collected from the bile ducts of infected rabbits 5 weeks post-infection. Excretory-secretory products (ESPs) and crude antigens of adult worms were prepared as previously described [28]. A total of 151 human sera were used in this study, 59 serum samples were collected from patients with clonorchiasis (egg-positive as detected by the KK and PCR method), and healthy individuals without parasitic infections (n = 74), 9 mixed infection sera (2 cases of mixed infection sera of C. sinensis and Schistosoma japonicum, 3 cases of C. sinensis and Trichinella spiralis, 2 cases of C. sinensis and Echinococcus granulosus and 2 cases of C. sinensis and Taenia solium), and sera from patients with other parasite infections (n = 9) (Schistosoma japonicum, Paragonimus westermani, Taenia solium, Trichuris trichura, Echinococcus granulosus, Ascaris lumbricoides, Ancylostoma duodenale, Trichinella spiralis and Toxoplasma gondii) were obtained from Chinese Center for Disease Control, and stored in our institute.…”

A novel Trichinella spiralis serine proteinase disrupted gut epithelial barrier and mediated larval invasion through binding to RACK1 and activating MAPK/ERK1/2 pathway

“…The results of GST pull-down analysis were mutually validated with Co-IP test. To verify whether rTsSPc is able to bind to RACK1 in Caco-2 cells, Co-IP was performed as previously reported [50][51][52]. In brief, 20 μg/ml rTsSPc was first incubated with Caco-2 cells at 37 ˚C for 2 h, and soluble cellular proteins were prepared with DISC lysis buffer (30 mM pH8.0 Tris-HCl, 120 mM NaCl, 10% glycerol, 1% TritonX-100).…”