Extracellular vesicles from Trichinella spiralis: Proteomic analysis and protective immunity

Trichinella spiralis (T. spiralis) derived extracellular vesicles (EVs) have been proposed to play a key role in regulating the host immune responses. In this study, we provided the first investigation of EVs proteomics released by T. spiralis muscle larvae (ML). T. spiralis ML EVs (Ts-ML-EVs) were successfully isolated and characterized by transmission electron microscopy (TEM) and western blotting. Using liquid chromatograph mass spectrometer (LC-MS/MS) analysis, we identified 753 proteins in the Ts-ML-EVs proteome and annotated by gene ontology (GO). These proteins were enriched in different categories by GO, kyoto encyclopedia of genes and genomes (KEGG) and domain analysis. GO enrichment analysis indicated association of protein deglutathionylation, lysosomal lumen and serine-type endopeptidase inhibitor activity with proteins which may be helpful during parasite-host interaction. Moreover, KEGG enrichment analysis revealed involvement of Ts-ML-EVs proteins in other glycan degradation, complement and coagulation cascades, proteasome and various metabolism pathways. In addition, BALB/c mice were immunized by subcutaneous injection of purified Ts-ML-EVs. Ts-ML-EVs group demonstrated a 23.4% reduction in adult worms and a 43.7% reduction in ML after parasite challenge. Cellular and humoral immune responses induced by Ts-ML-EVs were detected, including the levels of specific antibodies (IgG, IgM, IgE, IgG1 and IgG2a) as well as cytokines (IL-12, IFN-γ, IL-4 and IL-10) in serum. The results showed that Ts-ML-EVs could induce a Th1/Th2 mixed immune response with Th2 predominant. This study revealed a potential role of Ts-ML-EVs in T. spiralis biology, particularly in the interaction with host. This work provided a critical step to against T. spiralis infection based on Ts-ML-EVs.     

Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach

Although it has been known for many years that Trichinella spiralis initiates infection by invading intestinal epithelium, the mechanisms by which the parasite invades the intestinal epithelium are unknown. The purpose of this study was to screen the invasion-related proteins among the increased proteins of intestinal epithelial cells after culture with T. spiralis and to study their molecular functions. The proteins of HCT-8 cells which cultured with T. spiralis infective larvae were analyzed by SDS-PAGE and Western blot. Results showed that compared with proteins of normal HCT-8 cells, four additional protein bands (115, 61, 35 and 24 kDa) of HCT-8 cells cultured with the infective larvae were recognized by sera of the mice infected with T. spiralis, which may be the invasion-related proteins released by the infective larvae. Three bands (61, 35 and 24 kDa) were studied employing shotgun LC-MS/MS. Total 64 proteins of T. spiralis were identified from T. spiralis protein database by using SEQUEST searches, of which 43 (67.2%) proteins were distributed in a range of 10-70 kDa, and 26 proteins (40.6%) were in the range of pI 5-6. Fifty-four proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 54 annotated proteins, 43 proteins (79.6%) had binding activity and 23 proteins (42.6%) had catalytic activity (e.g. hydrolase, transferase, etc.), which might be related to the invasion of intestinal epithelial cells by T. spiralis. The protein profile provides a valuable basis for further studies of the invasion-related proteins of T. spiralis.     

Trichinella spiralis: comparison of stages in host intestine with those of an Arctic Trichinella sp

The intestinal phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic were compared in experimental animals. Reproductive capacity, pathogenicity, distribution, and persistence of adults in the small intestine, morphological measurements, and release of newborn larvae in vitro were examined. Numerous passages of 40 days each for T. spiralis and the Trichinella sp. isolate in mice did not affect reproductive capacity, distribution of adults in the small intestine, and size of worms. Reproductive capacity index for T. spiralis, I = 151.27 ± 27.30 was significantly higher compared to the Trichinella sp. isolate index, I = 63.46 ± 19.34. The Trichinella sp. isolate was more pathogenic to mice and wild rodents compared to T. spiralis during the intestinal phase. Both parasites were located in the anterior part of the small intestine but the position of T. spiralis adults (P = 17.08) differed from position of the Trichinella sp. isolate adults (P = 23.46) in the small intestine. Intestinal phase of T. spiralis was longer (20 days) compared to the Trichinella sp. isolate (15 days) but sex ratios (:♂) were similar for both parasites. T. spiralis females released significantly higher numbers of larvae in vitro/24 hr compared to the Trichinella sp. isolate. Release of larvae was continuous during the intestinal phase and the average fecundity for T. spiralis was 335 larvae/female and for the Trichinella sp. isolate 114 larvae/female. Adults of T. spiralis and the Trichinella sp. isolate were morphologically indistinguishable and did not differ in size. A comparative index for the intestinal phase is proposed for comparison of any Trichinella spp. isolates and standard T. spiralis.     

Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis

A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655 bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis.     

Characterisation of a Plancitoxin-1-Like DNase II Gene in Trichinella spiralis

Background

Deoxyribonuclease 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. {"type":"entrez-nucleotide","attrs":{"text":"XM_003370715.1","term_id":"339256039","term_text":"XM_003370715.1"}}XM_003370715.1) contains the HKD motif in its C-terminus domain.

Methodology/Principal Findings

In 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 {"type":"entrez-nucleotide","attrs":{"text":"KF984291","term_id":"594551321","term_text":"KF984291"}}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.

Conclusions

This 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.     

Therapeutic Potential of Myrrh and Ivermectin against Experimental Trichinella spiralis Infection in Mice

Trichinosis is a parasitic zoonosis caused by the nematode Trichinella spiralis. Anthelmintics are used to eliminate intestinal adults as well as tissue-migrating and encysted larvae. This study aimed to investigate the effects of ivermectin and myrrh obtained from the aloe-gum resin of Commiphora molmol on experimental trichinosis. Ninety albino mice were orally infected with 300 T. spiralis larvae. Drugs were tested against adult worms at day 0 and day 5 and against encysted larvae on day 15 and day 35 post-infection (PI). Mature worms and encysted larvae were counted in addition to histopathological examination of muscle specimens. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, globulin, urea, and creatinine values were estimated. Significant reductions in mean worm numbers were detected in ivermectin treated mice at day 0 and day 5 PI achieving efficacies of 98.5% and 80.0%, while efficacies of myrrh in treated mice were 80.7% and 51.5%, respectively. At days 15 and 35 post-infection, ivermectin induced significant reduction in encysted larval counts achieving efficacies of 76.5% and 54.0%, respectively, while myrrh efficacies were 76.6% and 35.0%, respectively. AST, ALT, urea, and creatinine levels were reduced, while total proteins were increased in response to both treatments compared to their values in the infected non-treated mice. Ivermectin use for controlling T. spiralis could be continued. Myrrh was effective and could be a promising drug against the Egyptian strains of T. spiralis with results nearly comparable to ivermectin.     

Trichinella spiralis: Comparison with an Arctic isolate

The muscle phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic was compared in experimental and wild animals. Reproductive capacity indices (RCI) of the Trichinella sp. isolate were significantly lower in laboratory rodents but were similar to T. spiralis in wild rodents. Sprague-Dawley rats were the most refractory to the Trichinella sp. isolate of all laboratory rodents. Outbred strains of mice were more susceptible to both T. spiralis and the Trichinella sp. isolate than inbred strains of mice. T. spiralis muscle larvae survived longer in mice and the survival of both T. spiralis and the Trichinella sp. isolate larvae was higher in female mice. While single pair interbreeding experiments showed reproductive isolation between T. spiralis and the Trichinella sp. isolate, multiple pair and transplant breeding experiments showed reproductive compatibility. Male and female infective larvae of T. spiralis and the Trichinella sp. isolate differed morphometrically, but a convergence in size of worms was observed after prolonged passages of the parasites in mice. Passaging history of the isolate and host species was found to have a significant effect on Trichinella morphology. It is proposed that the Trichinella sp. isolate is a physiological variant of T. spiralis and not a distinct species.     

Trichinella spiralis: Immunization of rats with an antigen fraction enriched for allergenicity

Trichinella spiralis whole muscle larval extract was fractionated by gel filtration and anion-exchange chromatography, and the protein fractions were assayed for allergenicity by a footpad-swelling test in mice; IgE antibody levels in rats immunized with the fractions were determined by passive cutaneous anaphylaxis test in rats. By these methods, an allergenic fraction from T. spiralis was isolated. The fraction, F1-b, was shown to be monodisperse by analysis with SDS-PAGE, IEP, and isoelectric focusing, indicating that it is a single protein moiety with a molecular weight of approximately 45,000 and a pI of approximately 5.1. The Schiff-periodate test showed Fl-b to be a glycoprotein. Rats immunized with Fl-b had significantly fewer intestinal worms than did nonimmunized controls at 24 hr and 7 days after oral challenge with T. spiralis larvae.     

Characterisation of the Trichinella spiralis deubiquitinating enzyme, TsUCH37, an evolutionarily conserved proteasome interaction partner

Background

Trichinella spiralis is a zoonotic parasitic nematode that causes trichinellosis, a disease that has been identified on all continents except Antarctica. During chronic infection, T. spiralis larvae infect skeletal myofibres, severely disrupting their differentiation state.

Methodology and Results

An activity-based probe, HA-Ub-VME, was used to identify deubiquitinating enzyme (DUB) activity in lysate of T. spiralis L1 larvae. Results were analysed by immuno-blot and immuno-precipitation, identifying a number of potential DUBs. Immuno-precipitated proteins were subjected to LC/MS/MS, yielding peptides with sequence homology to 5 conserved human DUBs: UCH-L5, UCH-L3, HAUSP, OTU 6B and Ataxin-3. The predicted gene encoding the putative UCH-L5 homologue, TsUCH37, was cloned and recombinant protein was expressed and purified. The deubiquitinating activity of this enzyme was verified by Ub-AMC assay. Co-precipitation of recombinant TsUCH37 showed that the protein associates with putative T. spiralis proteasome components, including the yeast Rpn13 homologue ADRM1. In addition, the UCH inhibitor LDN-57444 exhibited specific inhibition of recombinant TsUCH37 and reduced the viability of cultured L1 larvae.

Conclusions

This study reports the identification of the first T. spiralis DUB, a cysteine protease that is putatively orthologous to the human protein, hUCH-L5. Results suggest that the interaction of this protein with the proteasome has been conserved throughout evolution. We show potential for the use of inhibitor compounds to elucidate the role of UCH enzymes in T. spiralis infection and their investigation as therapeutic targets for trichinellosis.     

Partially Protective Immunity Induced by a 20 kDa Protein Secreted by Trichinella spiralis Stichocytes

Background

Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis.

Methods and Findings

The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response.

Conclusion

The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis.     

Trichinella spiralis: Differential effect of host bile on the in vitro invasion of infective larvae into epithelial cells

The differential effect of fox and pig bile and its corresponding low molecular weight fraction (LMW) was investigated on the in vitro invasion of MDCK-AA7 epithelial cell monolayers by Trichinella spiralis muscle larvae. Seven invasion experiments were performed and a total of 274 cell monolayers were examined. Fox and pig raw bile at 1:10 and 1:20 dilution and their LMW fractions at 1:10 dilution activated T. spiralis larvae to invade the cell monolayers. In addition, fox raw bile caused significantly larger cell damage than pig raw bile at both dilutions. The area of cell damage was larger at 1:10 than at 1:20 dilution for both fox and pig raw bile (p < 0.05). On the other hand, there was no significant difference between the areas of cell damage caused by the LMW fractions of fox and pig bile. It is concluded that differences between host bile actions may account for differences in host susceptibility to T. spiralis.     

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.     

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.     

Inactivation of the complement anaphylatoxin C5a by secreted products of parasitic nematodes

Given the importance of the complement anaphylatoxins in cellular recruitment during infection, the ability of secreted products from larval stages of Brugia malayi and Trichinella spiralis to influence C5a-mediated chemotaxis of human peripheral blood granulocytes in vitro was examined. Secreted products from B. malayi microfilariae almost completely abolished chemotaxis. This inhibition was blocked by phenylmethylsulphonyl fluoride, indicating the presence of a serine protease, which was subsequently shown to cleave C5a. In contrast, secreted products from T. spiralis infective larvae showed modest inhibition of C5a-mediated granulocyte chemotaxis, and this was blocked by potato carboxypeptidase inhibitor, an inhibitor of several metallocarboxypeptidases. Adult and larval stages of both parasites were demonstrated to secrete carboxypeptidases which cleaved hippuryl-l-lysine and hippuryl-l-arginine, and the T. spiralis enzyme was partially characterised. The data are discussed with reference to inflammation in parasitic nematode infection.