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Intestinal larva invasion is a crucial step of Trichinella spiralis infection. Intestinal infective larvae (IIL) and their excretory/secretory proteins (ESP) interact with gut epithelium, which often results in gut epithelium barrier injuries. Previous studies showed when T. spiralis invaded intestinal epithelium cells, the IIL ESP disrupted the tight junctions (TJs) of Caco-2 monolayer, but the mechanism is not clear. The IIL ESP might cause gut epithelial apoptosis, weaken the gut barrier and aid the larval invasion. The aim of this study was to investigate whether T. spiralis IIL ESP participate in enterocyte apoptosis and disrupt gut epithelial barrier to promote the larval invasion. Cell viability was assessed by CCK-8 assay and the results showed that 200 μg/ml of IIL ESP incubated with Caco-2 cells for 18 h inhibited the Caco-2 cell viability. The results of trans-epithelial electrical resistance (TEER) and FITC-dextran showed that IIL ESP decreased the TEER, increased FITC-dextran flux in Caco-2 monolayer. qPCR, Western blot and immunofluorescence test (IFT) showed that IIL ESP decreased the mRNA and protein expression of TJs (ZO-1, E-cad, Occludin and Claudin-1). The IIL ESP-induced Caco-2 cell apoptosis was observed by DAPI, Hoechst 33358, TUNEL and Annexin V/PI staining. Besides, flow cytometry revealed an increasing apoptosis rate in Caco-2 cells after the IIL ESP treatment. qPCR and Western blot analysis indicated that IIL ESP activated caspases (Caspase 3, Caspase 9 and Caspase 8), up-regulated the pro-apoptotic factors (Bax and Cytochrome c) and down-regulated the anti-apoptosis molecule Bcl-2. Interestingly, pretreatment of Caco-2 cells with apoptosis inhibitor Z-VAD-FMK abrogated and recovered the barrier function of Caco-2 monolayer destroyed by IIL ESP. Furthermore, the Z-VAD-FMK pretreatment also impeded the in vitro larva invasion of Caco-2 monolayer. T. spiralis IIL ESP induced gut epithelial apoptosis, reduced the TJs expression, damaged gut epithelial integrity and barrier function, and promoted larval invasion. These findings provided a basis of further understanding the interaction mechanism between T. spiralis and host gut epithelium, and they were valuable to the development new prevention and therapeutic strategy of early T. spiralis infection.

Larval invasion of gut mucosa is a crucial procedure in Trichinella spiralis infection. Previous studies showed that T. spiralis excretory-secretory proteins (ESP) disrupted gut epithelial integrity and promoted larval invasion by inducing apoptosis. However, the key molecules of the ESPs involved in this process are unknown. A serine protease (TsSPc) in T. spiralis ESPs was identified to be capable of promoting larval invasion. The aim of this study was to investigate whether TsSPc induces gut epithelial apoptosis, disrupts the barrier function, promotes larval invasion, and elucidates its acting mechanism. The results of DAPI, TUNEL and Annexin V/PI staining, immunofluorescence test (IFT) and flow cytometry showed that rTsSPc triggered Caco-2 cell apoptosis. The results of qPCR and Western blot revealed that rTsSPc significantly up-regulated the expression of Caco-2 cell apoptosis-related proteins (Caspase 3, Caspase 9 and Caspase 8, Bax and Cytochrome c), activated the apoptosis pathway, and thereby induced Caco-2 apoptosis. rTsSPc bound and interacted with PGAM5 receptor in Caco-2 cells and induced apoptosis, reduced the tight junctions (TJs) expression levels and damaged the Caco-2 monolayer integrity and barrier function, and promoted larval invasion. When gut epithelial PGAM5 receptor and apoptosis were inhibited by PGAM5-specific siRNA, inhibitor (LFHP-1c) and apoptosis inhibitor (Z-VAD-FMK), trans-epithelial electrical resistance (TEER) and TJs expression were obviously increased, and intestinal permeability was evidently decreased. Additionally, larval invasion of Caco-2 monolayer was also decreased by siPGAM5 and inhibitor pretreatment. These findings indicated that inhibition of PGAM5 receptor and apoptosis prevented rTsSPc from damaging gut epithelial integrity and larval invasion. rTsSPc binding and interacting with PGAM5 triggered gut epithelial apoptosis, reduced the TJs expression and damaged gut barrier function, thereby mediated larval invasion. TsSPc might be a candidate vaccine target to interdict larval invasion and T. spiralis infection.

Astacins, a family of zinc metalloproteinases, are involved in invasion and tissue migration processes in a variety of parasites. An astacin-like proteinases have been detected in the excretory-secretory products (ESPs) of Trichinella spiralis ( T. spiralis ), zinc metalloproteinase Nas-14 (TsNas14), but its function in T. spiralis remains unclear. The primary objective of this research was to delineate the molecular characterization of TsNas14 and explore its potential to compromise the integrity of the intestinal barrier. Results showed that TsNas14 contains an Astacin domain and two ShK domains. It is highly conserved and has a consistent transcriptional expression pattern in the Trichinella genus. Quantitative results showed that the TsNas14 is transcribed and expressed during the whole life cycle, but that the expression level was highest in the adult worm (AW) stage. In the 3d AW stage, TsNas14 is mainly distributed on the stichosome, ovary, cuticle, and hypodermis, while in the 6d AW stage, it is only present on the cuticle. Gelatin zymography showed that the oligomerized rTsNas14 had the enzyme activity to degrade gelatin, and could be effectively inhibited by 1,10-Phenanthroline, indicating that it had the natural activity of metalloproteinases. In vitro experiments showed that rTsNas14 can down-regulate the expression of occludin and claudin-1 proteins of human colorectal adenocarcinoma (Caco-2) cells and improve the permeability of an intestinal barrier model. In addition, the direct incubation of rTsNas14 with claduin-1 showed that rTsNas14 could significantly degrade claudin-1. In vivo studies have demonstrated that inhibition of TsNas14 expression significantly impairs the infectivity of T. spiralis in mice, resulting in a decreased AW and muscle larvae burden. These findings suggest that TsNas14 plays a crucial role in T. spiralis intestinal invasion and may serve as a novel potential target for Trichinella vaccines or therapeutic interventions.

Trichinella spiralis adult worms and larvae parasitize respectively in small intestine and skeletal muscles of the same host, and larval invasion of gut mucosa is the pivotal step for T. spiralis infection. A T. spiralis serine proteinase (TsSPc) was identified in its intestinal infective larva (IIL) ES antigens. TsSPc is involved in larval invasion of gut epithelium, but the mechanism is not completely elucidated. The purpose of this study was to investigate the mechanism of TsSPc action in larval invasion of gut mucosa. The results of molecular docking, immunofluorescence assay (IFA), GST pull-down and co-immunoprecipitation (Co-IP) showed that rTsSPc specifically bound to cytokeratin 8 (CK8) receptor in Caco-2 cells and activated RhoA/ROCK1 signaling pathway, as demonstrated by the evidently increased expression levels of CK8, RhoA and ROCK1. The results of qPCR and Western blot analysis revealed that binding of rTsSPc with CK8 and activation of RhoA/ROCK1 pathway significantly decreased the expression levels of gut epithelial tight junctions (TJs, E-cad, Occludin and Claudin-1), and increased the paracellular permeability. Knocking down CK8 in Caco-2 cells and ROCK1 pathway inhibitor Y27632 obviously inhibited the activation of RhoA/ROCK1 pathway, abolished rTsSPc-decreased TJs expression, rTsSPc-increased paracellular permeability, and inhibited larval invasion of Caco-2 monolayers in vitro. When the mice were pretreated with CK8 inhibitor Dasatinib and ROCK1 pathway inhibitor Y27632, and then orally infected with T. spiralis larvae, the activation of CK8 and RhoA/ROCK1 was significantly suppressed, intestinal permeability and adult worm burdens were obviously decreased, and intestinal inflammation was also distinctly alleviated. The number of intestinal goblet cells, expression of mucins (Muc2 and Muc5ac) and inflammatory cytokines (IL-1β, TNF-α, IL-10 and TGF-β) were significantly reduced. TsSPc binding to CK8 receptor in gut epithelium activated RhoA/ROCK1 pathway, reduced TJs expression and disrupted gut epithelial integrity, therefore mediated larval invasion of host gut mucosa. TsSPc might be considered as a promising vaccine molecular target for intercepting T. spiralis invasion and infection.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T . spiralis and T . britovi recognized by Trichinella -infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti- Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T . spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T . britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T . spiralis and 18 for T . britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T . spiralis ; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T . britovi . Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4′-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction.

Trichinella spiralis has the unique ability to make itself “at home” by creating and hiding in a new type of cell in the host body that is the nurse cell. From this immunologically privileged place, the parasite orchestrates a long-lasting molecular cross talk with the host through muscle larvae excretory-secretory products (ES L1). Those products can successfully modulate parasite-specific immune responses as well as responses to unrelated antigens (either self or nonself in origin), providing an anti-inflammatory milieu and maintaining homeostasis. It is clear, based on the findings from animal model studies, that T. spiralis and its products induce an immunomodulatory network (which encompasses Th2- and Treg-type responses) that may allow the host to deal with various hyperimmune-associated disorders as well as tumor growth, although the latter still remains unclear. This review focuses on studies of the molecules released by T. spiralis, their interaction with pattern recognition receptors on antigen presenting cells, and subsequently provoked responses. This paper also addresses the immunomodulatory properties of ES L1 molecules and how the induced immunomodulation influences the course of different experimental inflammatory and malignant diseases.

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. 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. 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 expresses paramyosin (Ts-PMY) not only as a structural protein but also as an immunomodulatory protein to protect the worm from being attacked by host complement components. In this study, the functions of PMY in the viability and the growth development of T. spiralis were confirmed at the first time by silencing the gene function with RNA interference technique. To understand its functions in the viability of the worm, we used RNA interference to silence the expression of Ts-pmy mRNA and protein in the parasite. Significant silencing of Ts-pmy mRNA expression in larval and adult T. spiralis was achieved by siRNA and dsRNA through soaking and electroporation. Electroporation of T. spiralis larvae with 8 µM siRNA1743 or 100 ng/µl dsRNA-PF3 resulted in 66.3% and 60.4% decrease in Ts-pmy transcript and 52.0% and 64.7% decrease in Ts-PMY protein expression, respectively, compared with larvae treated with irrelevant control siRNA or dsRNA. Larvae treated with siRNA1743 displayed significant reduction in molting (40.8%) and serious surface damage as detected with SYTOX fluorescent staining. Infection of mice with larvae electroporated with Ts-pmy siRNA1743 resulted in 37.6% decrease in adult worm burden and 23.2% decrease in muscle larvae burden compared with mice infected with control siRNA-treated larvae. In addition, adult worms recovered from mice infected with siRNA-treated larvae released 24.8% less newborn larvae. It is the first time RNAi was used on T. spiralis to demonstrate that silencing PMY expression in T. spiralis significantly reduces the parasite's viability and infectivity, further confirming that Ts-PMY plays an important role in the survival of T. spiralis and therefore is a promising target for vaccine development.

BACKGROUND: The intestinal phase is the early invasion stage of Trichinella spiralis (T. spiralis), in which muscle larvae invade intestine epithelial cells and then develop into adult worms to breed newborn larvae. Thus, intestinal infective larvae are first exposed to the immune system of the host, and antigens from the worms may be the earliest marker in the diagnosis of trichinellosis and may contribute to vaccine development to prevent Trichinella infections in pigs. METHODS: A cDNA library of intestinal infective larvae of T. spiralis at 6 hours post infection (p.i.) was constructed and immunoscreened using serum collected from pigs that were infected with T. spiralis at 26 days p.i. T. spiralis cystatin-like protein (Ts-CLP) gene encoding a 45.9 kDa protein was cloned and expressed in Escherichia coli. The rabbit antisera were generated and used to determine the location of Ts-CLP in the parasite. Transcription levels of Ts-CLP in different developmental stages of T. spiralis were observed by RT-PCR. The potential application of recombinant Ts-CLP in diagnosis against T. spiralis infection was tested by ELISA. The immune protection of recombinant Ts-CLP protein against T. spiralis infection was evaluated in mice. RESULTS: Thirty-three positive clones were selected from cDNA library, among which 20 clones encoded the same novel cystatin-like protein (Ts-CLP). Immunolocalisation and real-time quantitative PCR revealed that native Ts-CLP was localised primarily to β-stichocytes and that the Ts-clp gene was transcribed and expressed in all developmental stages of T. spiralis. The recombinant protein rTs-CLP was recognised by pig antiserum as early as 15 days p.i., and could induce protective immunity in mice, with a 61.21% reduction in the number of muscle larvae. CONCLUSIONS: These data preliminarily suggested that Ts-CLP may play an important role in the early infection of T. spiralis and that recombinant Ts-CLP protein is a candidate antigen for diagnosis and vaccine development in Trichinella infections.