A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

BACKGROUND: Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. METHODOLOGY/PRINCIPAL FINDING: TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. CONCLUSIONS: TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

Application of a recombinant novel trypsin from Trichinella spiralis for serodiagnosis of trichinellosis.

BACKGROUND: The excretory/secretory (ES) antigen of Trichinella spiralis muscle larvae (ML) is currently the most widely used diagnostic antigen to detect T. spiralis infection. However, this antigen has certain drawbacks, such as a complicated ES antigen preparation process and lower sensitivity during the early phase of infection. The aim of this study was to investigate the features of a novel T. spiralis trypsin (TsTryp) and evaluate its potential diagnostic value for trichinellosis. METHODS: The TsTryp gene was cloned and recombinant TsTryp (rTsTryp) expressed. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed to confirm the antigenicity of rTsTryp. The expression pattern and distribution signature of TsTryp at various life-cycle stages of T. spiralis were analyzed by quantitative PCR, western blotting and the immunofluorescence test. An ELISA with rTsTryp and ML ES antigens was used to detect immunoglobulins G and M (IgG, IgM) in serum samples of infected mice, swine and humans. The seropositive results were further confirmed by western blot with rTsTryp and ML ES antigens. RESULTS: TsTryp expression was observed in diverse T. spiralis life-cycle phases, with particularly high expression in the early developmental phase (intestinal infectious larvae and adults), with distribution observed mainly at the nematode outer cuticle and stichosome. rTsTryp was identified by T. spiralis-infected mouse sera and anti-rTsTryp sera. Natural TsTryp protease was detected in somatic soluble and ES antigens of the nematode. In mice infected with 200 T. spiralis ML, serum-specific IgG was first detected by rTsTryp-ELISA at 8 days post-infection (dpi), reaching 100% positivity at 12 dpi, and first detected by ES-ELISA at 10 dpi, reaching 100% positivity at 14 dpi. Specific IgG was detected by rTsTryp 2 days earlier than by ES antigens. When specific IgG was determined in serum samples from trichinellosis patients, the sensitivity of rTsTryp-ELISA and ES antigens-ELISA was 98.1% (51/52 samples) and 94.2% (49/52 samples), respectively (P = 0.308), but the specificity of rTsTryp was significantly higher than that of ES antigens (98.7% vs. 95.4%; P = 0.030). Additionally, rTsTryp conferred a lower cross-reaction, with only three serum samples in total testing positive from 11 clonorchiasis, 20 cysticercosis and 24 echinococcosis patients (1 sample from each patient group). CONCLUSIONS: TsTryp was shown to be an early and highly expressed antigen at intestinal T. spiralis stages, indicating that rTsTryp represents a valuable diagnostic antigen for the serodiagnosis of early Trichinella infection.

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

BACKGROUND: Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. METHODOLOGY/PRINCIPAL FINDING: IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. CONCLUSIONS: rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection.

Trichinella-derived protein ameliorates colitis by altering the gut microbiome and improving intestinal barrier function.

BACKGROUND: Inflammatory bowel disease (IBD) encompasses Crohn's Disease and Ulcerative Colitis. Reports have highlighted the potential use of helminths or their byproducts as a possible treatment for IBD; however, the mechanisms underlying their ability to modulate inflammation remain incompletely understood. In the present study, we analyze the possible mechanism of a serine protease inhibitor from adult T. spiralis excretion-secretion products (rTsSPI) on the improvement of colitis. METHODS: The immune protective effect of rTsSPI was studied by using DSS or Salmonella-induced colitis in female C56BL/6 mice. The effect of rTsSPI on the immune and inflammatory responses, gut microbiota, permeability of colon epithelium and junction proteins was analyzed. RESULTS: Treating mice with rTsSPI induced type 2 immunity and significantly attenuated clinical symptoms, macroscopical and histological features of DSS or bacteria-induced colonic inflammation. This was accompanied by decreasing neutrophil recruitment in the colonic lamina propria, and reducing TNF-α mRNA levels in the colon; in contrast, the recruitment of M2 macrophages, the expression level of IL-10 and adhesion molecules increased in the colon tissue. Moreover, treatment with rTsSPI led to an improvement in gut microbiota diversity, as well as an increase in the abundance of the bacterial genera Bifidobacterium and Ruminclostridium 5. CONCLUSIONS: Collective findings suggest that pretreatment with rTsSPI can ameliorate colitis in mice by inducing a Th2-type response with M2 macrophages. Data also indicate that immunotherapy with rTsSPI represents an additional strategy to ameliorate inflammatory processes in IBD by enhancing probiotic colonization and maintaining intestinal epithelial barrier function.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

BACKGROUND: Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. CONCLUSIONS: rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

The protective immunity induced by Trichinella spiralis galectin against larval challenge and the potential of galactomannan as a novel adjuvant.

Previous studies showed that recombinant Trichinella spiralis galectin (rTsgal) promoted larval invasion of gut epithelial cells, while anti-rTsgal antibodies inhibited the invasion. Galactomannan (GM) is a polysaccharide capable of regulating immune response. The aim of this study was to evaluate protective immunity induced by rTsgal immunization and the potential of GM as a novel adjuvant. The results showed that vaccination of mice with rTsgal+ISA201 and rTsgal+GM elicited a Th1/Th2 immune response. Mice immunized with rTsgal+ISA201 and rTsgal+GM exhibited significantly higher levels of serum anti-rTsgal antibodies, mucosal sIgA and cellular immune responses, but level of specific antibodies and cytokines of rTsgal+GM group was lower than the rTsgal+ISA201 group. Immunization of mice with rTsgal+ISA201 and rTsgal+GM showed a 50.5 and 40.16% reduction of intestinal adults, and 52.04 and 37.53% reduction of muscle larvae after challenge. Moreover, the numbers of goblet cells and expression level of mucin 2, Muc5ac and pro-inflammatory cytokines (TNF-α and IL-1ß) in gut tissues of vaccinated mice were obviously decreased, while Th2 inducing cytokine (IL-4) expression was evidently increased. Galactomannan enhanced protective immunity, alleviated intestinal and muscle inflammation of infected mice. The results indicated that rTsgal+ISA201 vaccination induced a more prominent gut local as well as systemic immune response and protection compared to rTsgal+GM vaccination. The results suggested that Tsgal could be considered as a candidate vaccine target against Trichinella infection and galactomannan might be a potential novel candidate adjuvant of anti-Trichinella vaccines.

Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of newborn larvae.

BACKGROUND: During the early stages of Trichinella spiralis infection, macrophages predominantly undergo polarization to the M1-like phenotype, causing the host's inflammatory response and resistance against T. spiralis infection. As the disease progresses, the number of M2-type macrophages gradually increases, contributing to tissue repair processes within the host. While cysteine protease overexpression is typically associated with inflammation, the specific role of T. spiralis cathepsin L (TsCatL) in mediating macrophage polarization remains unknown. The aim of this study was to assess the killing effect of macrophage polarization mediated by recombinant T. spiralis cathepsin L domains (rTsCatL2) on newborn larvae (NBL). METHODS: rTsCatL2 was expressed in Escherichia coli strain BL21. Polarization of the rTsCatL2-induced RAW264.7 cells was analyzed by enzyme-linked immunosorbent assay (ELISA), quantitative PCR (qPCR), western blot, immunofluorescence and flow cytometry. The effect of JSH-23, an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), on rTsCatL2-induced M1 polarization investigated. Cytotoxic effects of polarized macrophages on NBL were observed using in vitro killing assays. RESULTS: Following the co-incubation of rTsCatL2 with RAW264.7 murine macrophage cells, qPCR and ELISA revealed increased transcription and secretion levels of inducible nitric oxide synthase (iNOS), interleukin (IL)-6, IL-1ß and tumor necrosis factor alpha (TNF-α) in macrophages. Western blot analysis showed a significant increase in iNOS protein expression, while the expression level of arginase-1 protein remained unchanged. Flow cytometry revealed a substantial increase in the number of CD86-labeled macrophages. The western blot results also indicated that rTsCatL2 increased the expression levels of phospho-NF-κB and phospho-nuclear factor-κB inhibitor alpha (IκB-α) proteins in a dose-dependent manner, while immunofluorescence revealed that rTsCatL2 induced nuclear translocation of the p65 subunit of NF-κB (NF-κB p65) protein in macrophages. The inhibitory effect of JSH-23 suppressed and abrogated the effect of rTsCatL2 in promoting M1 macrophage polarization. rTsCatL2 mediated polarization of macrophages to the M1-like phenotype and enhanced macrophage adhesion and antibody-dependent cell-mediated cytotoxicity (ADCC) killing of NBL. CONCLUSIONS: The results indicated that rTsCatL2 induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of NBL. This study provides a further understanding of the interaction mechanism between T. spiralis and the host.

Binding of Trichinella spiralis C-type lectin with syndecan-1 on intestinal epithelial cells mediates larval invasion of intestinal epithelium.

C-type lectin (CTL) is a protein that binds to saccharides and plays an important role in parasite adhesion, host cell invasion and immune evasion. Previous studies showed that recombinant T. spiralis C-type lectin (rTsCTL) promotes larval invasion of intestinal epithelium cells (IEC), whereas anti-rTsCTL antibodies inhibits larval invasion. Syndecan-1 (SDC-1) is a member of the heparan sulfate proteoglycan family which is mainly expressed on the surface of IEC and in extracellular matrices where they interact with a plethora of ligands. SDC-1 has a principal role in maintaining cell morphogenesis, establishing cell-cell adhesions, and regulating the gut mucosal barrier. The aim of this study was to investigate whether rTsCTL binds to SDC-1 on IEC, and the binding of rTsCTL with SDC-1 promotes larval invasion and its mechanism. IFA results show that rTsCTL and SDC-1 co-localized on Caco-2 cell membrane. GST pull-down and Co-IP verified the direct interaction between rTsCTL and SDC-1 on Caco-2 cells. qPCR and Western blotting revealed that rTsCTL binding to SDC-1 increased the expression of SDC-1 and claudin-2, and reduced the expression of occludin and claudin-1 in Caco-2 cells incubated with rTsCTL via the STAT3 pathway. ß-Xyloside (a syndecan-1 synthesis inhibitor) and Stattic (a STAT3 inhibitor) significantly inhibited rTsCTL binding to syndecan-1 in Caco-2 cells and activation of the STAT3 pathway, abrogated the effects of rTsCTL on the expression of gut tight junctions, and impeded larval invasion. The results demonstrate that binding of rTsCTL to SDC-1 on Caco-2 cells activated the STAT3 pathway, decreased gut tight junction expression, damaged the integrity of the gut epithelial barrier, and mediated T. spiralis invasion of the gut mucosa. TsCTL might be regarded as a candidate vaccine target against T. spiralis invasion and infection.

Trichinella spiralis dipeptidyl peptidase 1 suppressed macrophage cytotoxicity by promoting M2 polarization via the STAT6/PPARγ pathway.

Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1), or cysteine cathepsin C, is a secretory protein that is highly expressed during the infective larvae and adult worm stages in the intestines. The aim of this study was to investigate the mechanism by which recombinant TsDPP1 (rTsDPP1) activates macrophages M2 polarization and decreases macrophage cytotoxicity to kill newborn larvae via ADCC. RAW264.7 macrophages and murine peritoneal macrophages were used in this study. The results of the immunofluorescence test (IFT) and confocal microscopy showed that rTsDPP1 specifically bound to macrophages, and the binding site was localized on the cell membrane. rTsDPP1 activated macrophage M2 polarization, as demonstrated by high expression levels of Arg1 (M2 marker) and M2-related genes (IL-10, TGF-ß, CD206 and Arg1) and high numbers of CD206+ macrophages. Furthermore, the expression levels of p-STAT6, STAT6 and PPARγ were obviously increased in rTsDPP1-treated macrophages, which were evidently abrogated by using a STAT6 inhibitor (AS1517499) and PPARγ antagonist (GW9662). The results indicated that rTsDPP1 promoted macrophage M2 polarization through the STAT6/PPARγ pathway. Griess reaction results revealed that rTsDPP1 suppressed LPS-induced NO production in macrophages. qPCR and flow cytometry results showed that rTsDPP1 downregulated the expression of FcγR I (CD64) in macrophages. The ability of ADCC to kill newborn larvae was significantly decreased in rTsDPP1-treated macrophages, but AS1517499 and GW9662 restored its killing capacity. Our results demonstrated that rTsDPP1 induced macrophage M2 polarization, upregulated the expression of anti-inflammatory cytokines, and inhibited macrophage-mediated ADCC via activation of the STAT6/PPARγ pathway, which is beneficial to the parasitism and immune evasion of this nematode.

Application of molecular markers in the research of genetic diversity in medical helminths / 中国热带医学

@#Human-animal parasitic diseases caused by medical helminths are hazardous to human health. Genetic polymorphism studies on medical helminth populations can not only understand the biological characteristics and genetic structure of their populations, but also help reveal how they adapt to their parasitic environment, thus contributing to deepen our understanding of the epidemiological patterns of parasitic diseases and improve our understanding of accurate prevention and control of parasitic diseases. With the development of molecular biology, molecular markers such as DNA barcodes, simple sequence repeats, and single nucleotide polymorphism markers have been widely used to study the genetic relationships among parasite populations and individuals, and to reveal the genetic variation of parasite populations and the evolution of species origins. In this paper, we systematically review the application of three molecular markers commonly used in the study of genetic polymorphism in medical helminths, with a view to laying the foundation for related research.

Cloning and Expression of a New Trichinella spiralis Serine Protease and Its Role in Invading Host Intestinal Epithelium.

Background: In previous studies, a new Trichinella spiralis serine protease 1.1 (TsSP1.1) was identified in surface proteins of T. spiralis muscle larvae (ML) by proteomics analysis, but its functions in T. spiralis infection are unknown. The aim of this study was to investigate the roles of TsSP1.1 during larval intrusion of gut epithelium. Methods: From January 2019 to March 2021, complete TsSP1.1 cDNA sequence was cloned and expressed in Escherichia coli BL21 at the Department of Parasitology, Medical College of Zhengzhou University, Zhengzhou, China. Expression and location of TsSP1.1 in the parasite were investigated using indirect immunofluorescence assay (IIFA) and Western blotting. The in vitro intestinal epithelium cells (IECs) intrusion assay was used to ascertain the roles of TsSP1.1 during larval intrusion of IECs and gut epithelium. Results: TsSP1.1 was a surface and secretory protein, which was expressed at various T. spiralis stages, and principally localized at cuticle, stichosome and embryos of the nematode. rTsSP1.1 accelerated larval intrusion of IECs, whereas anti-rTsSP1.1 antibodies impeded larval intrusion. The acceleration and inhibtion was dose-dependently related to rTsSP1.1 and anti-TsSP1.1 antibodies. Block of the IIL with anti-rTsSP1.1 serum also impeded larval intrusion of gut mucosa. Conclusion: TsSP1.1 participates in T. spiralis intrusion of gut epithelium.

Oral immunization of mice with recombinant Lactobacillus plantarum expressing a Trichinella spiralis galectin induces an immune protection against larval challenge.

BACKGROUND: Trichinella spiralis is an important foodborne parasite that presents a severe threat to food safety. The development of an anti-Trichinella vaccine is an important step towards controlling Trichinella infection in food animals and thus ensure meat safety. Trichinella spiralis galectin (Tsgal) is a novel protein that has been identified on the surface of this nematode. Recombinant Tsgal (rTsgal) was found to participate in larval invasion of intestinal epithelium cells (IECs), whereas anti-rTsgal antibodies impeded the invasion. METHODS: The rTsgal/pSIP409- pgsA' plasmid was constructed and transferred into Lactobacillus plantarum strain NC8, following which the in vitro biological properties of rTsgal/NC8 were determined. Five groups of mice were orally immunized three times, with a 2-week interval between immunizations, with recombinant NC8-Tsgal, recombinant NC8-Tsgal + α-lactose, empty NC8, α-lactose only or phosphate-buffered saline (PBS), respectively. The vaccinated mice were infected orally with T. spiralis larvae 2 weeks following the last vaccination. Systemic and intestinal local mucosal immune responses and protection were also assessed, as were pathological changes in murine intestine and skeletal muscle. RESULTS: rTsgal was expressed on the surface of NC8-Tsgal. Oral immunization of mice with rTsgal vaccine induced specific forms of serum immunoglobulin G (IgG), namely IgG1/IgG2a, as well as IgA and gut mucosal secretion IgA (sIgA). The levels of interferon gamma and interleukin-4 secreted by cells of the spleen, mesenteric lymph nodes, Peyer's patches and intestinal lamina propria were significantly elevated at 2-6 weeks after immunization, and continued to rise following challenge. Immunization of mice with the oral rTsgal vaccine produced a significant immune protection against T. spiralis challenge, as demonstrated by a 57.28% reduction in the intestinal adult worm burden and a 53.30% reduction in muscle larval burden, compared to the PBS control group. Immunization with oral rTsgal vaccine also ameliorated intestinal inflammation, as demonstrated by a distinct reduction in the number of gut epithelial goblet cells and mucin 2 expression level in T. spiralis-infected mice. Oral administration of lactose alone also reduced adult worm and larval burdens and relieved partially inflammation of intestine and muscles. CONCLUSIONS: Immunization with oral rTsgal vaccine triggered an obvious gut local mucosal sIgA response and specific systemic Th1/Th2 immune response, as well as an evident protective immunity against T. spiralis challenge. Oral rTsgal vaccine provided a prospective approach for control of T. spiralis infection.

Oral vaccination of mice with attenuated Salmonella encoding Trichinella spiralis calreticulin and serine protease 1.1 confers protective immunity in BALB/c mice.

BACKGROUND: Trichinella spiralis is a foodborne parasitic nematode which is a serious risk to meat safety. Development of anti-Trichinella vaccine is needed to control Trichinella infection in food animals. In this study, two novel T. spiralis genes (calreticulin and serine protease 1.1) in combination were used to construct oral DNA vaccines, and their induced protective immunity was evaluated in a murine model. METHODOLOGY/PRINCIPAL FINDINGS: TsCRT+TsSP1.1, TsCRT and TsSP1.1 DNA were transformed into attenuated Salmonella typhimurium ΔcyaSL1344. Oral vaccination of mice with TsCRT+TsSP1.1, TsCRT and TsSP1.1 DNA vaccines elicited a gut local mucosal sIgA response and systemic Th1/Th2 mixed response. Oral vaccination with TsCRT+TsSP1.1 induced obviously higher level of serum specific antibodies, mucosal sIgA and cellular immune response than either of single TsCRT or TsSP1.1 DNA vaccination. Oral vaccination of mice with TsCRT+TsSP1.1 exhibited a 53.4% reduction of enteral adult worms and a 46.05% reduction of muscle larvae, conferred a higher immune protection than either of individual TsCRT (44.28 and 42.46%) or TsSP1.1 DNA vaccine (35.43 and 29.29%) alone. Oral vaccination with TsCRT+TsSP1.1, TsCRT and TsSP1.1 also obviously ameliorated inflammation of intestinal mucosa and skeletal muscles of vaccinated mice after challenge. CONCLUSIONS: TsCRT and TsSP1.1 might be regarded the novel potential targets for anti-Trichinella vaccines. Attenuated Salmonella-delivered DNA vaccine provided a prospective approach to control T. spiralis infection in food animals.

A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells.

The aim of this study was to investigate the characteristics of a novel type C lectin from Trichinella spiralis (TsCTL) and its role in larval invasion of intestinal epithelial cells (IECs). TsCTL has a carbohydrate recognition domain (CRD) of C-type lectin. The full-length TsCTL cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of qPCR, Western blotting and immunofluorescence assays (IFAs) showed that TsCTL was a surface and secretory protein that was highly expressed at the T. spiralis intestinal infective larva (IIL) stages and primarily located at the cuticle, stichosome and embryos of the parasite. rTsCTL could specifically bind with IECs, and the binding site was localized in the IEC nucleus and cytoplasm. The IFA results showed that natural TsCTL was secreted and bound to the enteral epithelium at the intestinal stage of T. spiralis infection. The rTsCTL had a haemagglutinating effect on murine erythrocytes, while mannose was able to inhibit the rTsCTL agglutinating effect for mouse erythrocytes. rTsCTL accelerated larval intrusion into the IECs, whereas anti-rTsCTL antibodies and mannose significantly impeded larval intrusion in a dose-dependent manner. The results indicated that TsCTL specifically binds to IECs and promotes larval invasion of intestinal epithelium, and it might be a potential target of vaccines against T. spiralis enteral stages.

Characterization of a novel pyruvate kinase from Trichinella spiralis and its participation in sugar metabolism, larval molting and development.

BACKGROUND: Pyruvate kinase widely exists in many parasites and plays an important role in the energy production for the parasites. Pyruvate kinase might be a potential drug target for killing the parasites. The aim of the present study was to evaluate the biological characteristics and roles of T. spiralis pyruvate kinase M (TsPKM) in sugar metabolism, larval molting and development of T. spiralis. METHODOLOGY/PRINCIPAL FINDINGS: TsPKM has two functional domains of pyruvate kinase and the tertiary structure of TsPKM is tetramer which has the enzyme active site constituted by 8 amino-acid residues (Arg71, Asn73, Asp110, Phe241, Lys267, Glu269, Asp293 and Thr325). Recombinant TsPKM (rTsPKM) was expressed and purified. The rTsPKM had good immunogenicity. RT-PCR and Western blot showed that TsPKM was transcribed and expressed at various developmental stages in T. spiralis lifecycle. Immunofluorescence test showed that TsPKM was principally located in the cuticle, muscle, stichosome, intestine and the intrauterine embryos of female adults. rTsPKM catalyzed the reaction of phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to produce pyruvic acid and adenosine triphosphate (ATP). TsPKM played an important role in the metabolism and energy production of T. spiralis. After silencing of TsPKM gene by specific dsRNA-TsPKM2, protein expression and enzyme activity of TsPKM decreased by 50.91 and 26.06%, respectively. After treatment with RNAi, natural TsPKM enzyme activity, larval molting, sugar metabolism, growth and development of T. spiralis were significantly reduced. CONCLUSIONS: TsPKM participates in the larval molting, sugar metabolism, growth and development of T. spiralis and it might be a candidate target of therapeutic drug of trichinellosis.

Characterization of a novel aminopeptidase P from Trichinella spiralis and its participation in the intrusion of intestinal epithelial cells.

Aminopeptidases P are metalloproteases belonging to the M24 peptidase family. It specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids, and plays an important role in the nutrition, metabolism and growth of parasites. The aim of this study was to characterize a novel Trichinella spiralis aminopeptidase P (TsAPP) and to investigate its functions in the invasion of T. spiralis. TsAPP contained two domains of creatinase (a creatinase N and creatinase N2) and a domain of peptidase M24C and APP. The complete TsAPP sequence was cloned and expressed in Escherichia coli BL21 cells. The recombinantly produced TsAPP was used to raise polyclonal antibodies that were subsequently used to detect the expression of the protein in the different life stages of T. spiralis. TsAPP was expressed in various T. spiralis stages. TsAPP was primarily localized in the cuticle, stichosome and intrauterine embryos of this nematode. rTsAPP has an enzymatic activity of a natural aminopeptidase P to hydrolyze the substrate H-Ala-Pro-OH. rTsAPP promoted the larval intrusion of intestinal epithelium cells (IECs). The results showed that TsAPP is involved in the T. spiralis intrusion of IECs and it might be a potential candidate vaccine target against Trichinella infection.

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis.

Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 µM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.

Preexisting Trichinella spiralis infection attenuates the severity of Pseudomonas aeruginosa-induced pneumonia.

BACKGROUND: A range of helminth species involve the migration of developing larvae through the lung and establish chronic infections in the host that include potent immune regulatory effects. Trichinella spiralis is one of the most successful parasitic symbiotes. After released by intestinal female adult worms, newborn larvae of T. spiralis travel through the circulatory system to the lung and finally reach skeletal muscle cells. As unique inflammation modulator of intracellular parasitism, T. spiralis shows improved responses to autoimmune disease and viral pulmonary inflammation by exerting immunomodulatory effects on innate and adaptive immune cells. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 mice were divided into four groups: uninfected; helminth- T. spiralis infected; P. aeruginosa infected; and co-infected. Mice infected with T. spiralis were incubated for 6 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage fluid, blood and lung samples were analyzed. We found that T. spiralis induced Th2 response in the mouse lung tissue, increased lung CD4+ T cells, GATA3, IL-4, IL-5 and IL-13 expression. Pre-existing T. spiralis infection decreased lung neutrophil recruitment, inflammatory mediator IL-1ß and IL-6 expression and chemokine CXCL1 and CXCL2 release during P. aeruginosa- pneumonia. Furthermore, T. spiralis co-infected mice exhibited significantly more eosinophils at 6 hours following P. aeruginosa infection, ameliorated pulmonary inflammation and improved survival in P. aeruginosa pneumonia. CONCLUSIONS: These findings indicate that a prior infection with T. spiralis ameliorates experimental pulmonary inflammation and improves survival in P. aeruginosa pneumonia through a Th2-type response with eosinophils.

Proteases secreted by Trichinella spiralis intestinal infective larvae damage the junctions of the intestinal epithelial cell monolayer and mediate larval invasion.

The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development.

Characterization of a Novel Glutamine Synthetase From Trichinella spiralis and Its Participation in Larval Acid Resistance, Molting, and Development.

Trichinella spiralis is a major foodborne parasite worldwide. After the encapsulated muscle larvae (ML) in meat are ingested, the ML are liberated in the stomach of the host and activated into intestinal infectious larvae (IIL), which develop into adult worm after molting four times. A novel glutamine synthetase (TsGS) was identified from T. spiralis IIL at 10 h post-infection, but its biological role in T. spiralis life cycle is not clear. The aim of this study was to investigate the biological characteristics of TsGS and its functions in larval acid resistance, molting, and development. TsGS has a glutamine synthetase (GS) catalytic domain. Complete TsGS sequence was cloned and expressed in Escherichia coli BL21. rTsGS has good immunogenicity. qPCR and Western blotting showed that TsGS was highly expressed at IIL stage, and immunofluorescence revealed that TsGS was principally localized at the cuticle and intrauterine embryos of this nematode. rTsGS has enzymatic activity of natural GS to hydrolyze the substrate (Glu, ATP, and NH4 +). Silencing of TsGS gene significantly reduced the IIL survival at pH 2.5, decreased the IIL burden, and impeded larval molting and development. The results demonstrated that TsGS participates in T. spiralis larval acid resistance, molting and development, and it might be a candidate vaccine target against Trichinella molting and development.