Ge Ge Sun

Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae.

The critical step for Trichinella spiralis infection is that muscle larvae (ML) are activated to intestinal infective larvae (IIL) and invade intestinal epithelium to further develop. The IIL is its first invasive stage, surface proteins are directly exposed to host environment and are crucial for larval invasion and development. In this study, shotgun LC-MS/MS was used to analyze surface protein profiles of ML and IIL. Totally, 41 proteins common to both larvae, and 85 ML biased and 113 IIL biased proteins. Some proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Gene ontology analysis revealed that proteins involved in generation of precursor metabolites and energy; and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process were enriched in IIL at level 4. Some IIL biased proteins might play important role in larval invasion and development. qPCR results confirmed the high expression of some genes in IIL. Our study provides new insights into larval invasion, host-Trichinella interaction and for screening vaccine candidate antigens.

Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis

The Trichinella spiralis 31 kDa protein (Ts31) was screened from the excretory-secretory (ES) proteins of muscle larvae (ML) by immunoproteomics using serum from mice infected with T. spiralis at 18 days post infection (dpi). The aim of this study was to characterize the Ts31 protein and to evaluate the potential of the recombinant Ts31 protein (rTs31) for serodiagnosis of human trichinellosis. Ts31 gene was cloned and rTs31 was produced in an E. coli expression system. An anti-rTs31serum recognized the native protein migrating in a 25-55 kDa range by Western blotting of ML crude or ES antigens. Expression of Ts31 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and ML). An immunolocalization analysis identified Ts31 in the cuticle and stichocytes of the parasite. The sensitivity of rTs31-ELISA and ES antigen ELISA for detecting anti-Trichinella IgG antibodies in sera of patients with trichinellosis was 97.83% (45/46) and 86.78% (39/46), respectively (P>0.05); The specificity of rTs31-ELISA was 99.13% (114/115), which was significantly higher than 85.22% (98/115) of ES antigen ELISA (P<0.01). The rTs31 protein of T. spiralis could be considered as a potential diagnostic antigen for trichinellosis.

Trichinella spiralis: Low vaccine potential of glutathione S-transferase against infections in mice

We have previously reported that Trichinella spiralis glutathione-S-transferase (TsGST) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML). In this study, the TsGST gene was cloned, and recombinant TsGST (rTsGST) was produced. Anti-rTsGST serum recognized the native TsGST by Western blotting in crude antigens of ML, adult worm (AW) and newborn larvae (NBL) of T. spiralis, but not in ML excretory-secretory (ES) antigens. Expression of TsGST was observed in all different developmental stages (IIL, AW, NBL and ML). An immunolocalization analysis identified TsGST in the cuticle, stichosome and genital primordium of the parasite. The rTsGST had GST enzymatic activity. After a challenge infection with T. spiralis larvae, mice immunized with rTsGST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with rTsGST induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1) and partial protective immunity against T. spiralis infection.

Proteomic analysis of Trichinella spiralis adult worm excretory-secretory proteins recognized by early infection sera

At the intestinal stage of a Trichinella spiralis (T. spiralis) infection, the excretory-secretory (ES) antigens produced by adult worms (AWs) result in an early exposure to the hosts immune system and elicit the production of specific antibodies; the AW ES proteins might provide early diagnostic markers of trichinellosis. The aim of this study was to identify early serodiagnostic markers from T. spiralis AW ES antigens. T. spiralis AWs were collected at 72h post infection, and their ES antigens were analysed by SDS-PAGE and Western blot. Then, the immunoreactive bands were subjected to shotgun LC-MS/MS and bioinformatics analyses. Our results showed that only one protein band (33kDa) was recognized by the sera of mice infected with T. spiralis at 8 days after infection. The shotgun LC-MS/MS analysis identified 23 proteins that were then clustered into 10 types; these proteins had molecular weights of 28.13-71.62kDa and pI 5.05-9.20. Certain enzymes (e.g., serine protease, adult-specific deoxyribonuclease [DNase] II, peptidase S1A subfamily, and multi cystatin-like domain protein) were found to be highly represented. The functions of the 10 proteins were further analysed: of the 6 annotated proteins, 3 had serine hydrolase activity and 2 had DNase II activity. These results provide a valuable basis for identifying early diagnostic antigens and vaccine candidates for trichinellosis.

Characterization of a Trichinella spiralis putative serine protease. Study of its potential as sero-diagnostic tool

Background 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. Methodology/Principal findings 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. Conclusions The rTsSP is a potential early diagnostic antigen for human trichinellosis.

The Immune Protection Induced by a Serine Protease Inhibitor From the Foodborne Parasite Trichinella spiralis

Serine protease inhibitors (SPI) are a superfamily of the proteins able to suppress serine protease activity, and may exert the major biological function in complement activation, inflammation, and fibrinolysis. A SPI was identified from Trichinella spiralis adult worms (AW) by immunoproteomics with early infection sera. The aim of this study was to investigate the protective immune elicited by TsSPI. The complete TsSPI cDNA sequence was cloned into pQE-80 L and then expressed in Escherichia coli BL21. The rTsSPI was purified and its antigenicity was determined by Western blotting analysis. By using anti-rTsSPI serum the native TsSPI was identified in somatic and ES proteins from muscle larvae (ML). The results of qPCR and immunofluorescence assay (IFA) revealed that the expression of the TsSPI gene was observed throughout all developmental stages of T. spiralis (ML, intestinal infective larvale, 3- and 6-days AW, and newborn larvae, NBL), located principally in cuticles, stichosome, and embryos of this parasitic nematode. Vaccination of mice with rTsSPI triggered high level of anti-TsSPI IgG response, and showed a 62.2 and 57.25% worm burden reduction in the recovery of intestinal AW at 6 days post-infection (dpi) and ML at 35 dpi, respectively. The TsSPI might be a novel potential target for anti-Trichinella vaccine.

Cloning, expression and characterisation of a cysteine protease from Trichinella spiralis

Cysteine protease is a superfamily of widespread proteolytic enzymes and plays a major role in larval invasion, migration, exsheathing, survival and immune evasion in parasites. In the present study, the gene coding cysteine proteinase of the nematode Trichinella spiralis (Owen, 1835) was cloned into pQE-80L and subsequently expressed in E. coli JM109. The rTsCP was purified and its antigenicity was identified by Western blot and ELISA. Using anti-rTsCP serum the native TsCP was identified in muscle larval crude proteins. The results of quantitative real-time PCR and immunofluorescence test demonstrated that the TsCP was expressed in all stages of T. spiralis and located mainly in cuticle, stichosome and reproductive organs. The immunisation of mice with rTsCP elicited Th2-predominant immune responses. Anti-rTsCP antibodies could partially inhibit the in vitro larval invasion of intestinal epithelial cells and kill the newborn larvae by an antibody-dependent cell-mediated dose-dependent cytotoxicity. The vaccinated mice exhibited a 54% reduction of adults and a 33% reduction of muscle larvae following challenge infection. The results suggested that the TsCP might be an indispensable protein in Trichinella invasion, development and survival of T. spiralis in hosts, and could be a potential vaccine target against infection.

Characterization of a serine protease inhibitor from Trichinella spiralis and its participation in larval invasion of host’s intestinal epithelial cells

BackgroundTrichinella spiralis serine protease inhibitor (TsSPI) was identified in ES proteins of adult worms (AW), the TsSPI gene was highly expressed at enteral stage worms (AW and newborn larvae), distributed mainly in the cuticle and stichosome of this nematode. Vaccination of mice with rTsSPI exhibited a 62.2% reduction of intestinal AW and a 57.25% reduction of muscle larvae after larval challenge. The aim of this study was to investigate the biological characteristics of TsSPI and its roles in the process of T. spiralis invasion of host’s intestinal epithelium cells (IECs).MethodsThe rTsSPI inhibition on trypsin enzymatic activity was detected by SDS-PAGE and spectrophotometry. The binding of rTsSPI with intestinal epithelium from normal mice and the primary cultured mouse intestinal epithelium cells (IECs) was examined by indirect immunofluorescent (IIF), the cellular localization of rTsSPI binding to IECs was observed by confocal microscopy. The inhibition of anti-rTsSPI serum on T. spiralis invasion of IECs was determined by an in vitro invasion assay. Anti-rTsSPI antibody cytotoxicity on the newborn larvae (NBL) was also determined.ResultsThe rTsSPI had the inhibitory activity against porcine trypsin. The rTsSPI specifically bound to the intestinal epithelium from normal mice and primary cultured mouse IECs, and the binding sites were located in IEC membrane and cytoplasm. Anti-rTsSPI antibodies depressed the larval invasion of IECs with a dose-dependent mode. Anti-rTsSPI antibodies also participated in the destruction of T. spiralis NBL via an ADCC-mediated manner.ConclusionsTsSPI might participate in the T. spiralis larval invasion of IECs and it is likely the potential vaccine target against T. spiralis enteral stages.