Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Molecular characteristics of Echinococcus multilocularis FABP1 and its regulatory functions on murine macrophages.

Fatty acid binding proteins (FABPs) have emerged as attractive vaccination candidates for several platyhelminth species. To explore the physiological functions of Echinococcus multilocularis (E. multilocularis) FABP, the molecular characteristics of EmFABP1 were analyzed by online software, and the regulatory roles of rEmFABP1 protein in murine macrophages were further investigated. The emfabp1 gene encodes 133 amino acids with the characteristic ß-barrel shape of the cytoplasmic FABP family. Natural EmFABP1 protein is predominantly expressed in protoscoleces tegument and germinal layer cells and is also detected in cyst fluid and exosomes of E. multilocularis. rEmFABP1 protein demonstrated a notable suppression of phagocytic activity and nitric oxide production in murine macrophages. Additionally, the protein was observed to promote apoptosis and regulate cytokine expression in macrophages. These findings suggested that E. multilocularis FABP1 is critical in modifying macrophage physiological processes and that this protein may have immunomodulatory roles during infection.

The nematode effector Mj-NEROSs interacts with Rieske's iron-sulfur protein influencing plastid ROS production to suppress plant immunity.

Root-knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj-NEROSs) within the host plant environment. Mj-NEROSs suppresses INF1-induced cell death as well as flg22-induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS-related genes upon Mj-NEROSs expression. NEROSs interacts with the plant Rieske's iron-sulfur protein (ISP) as shown by yeast-two-hybrid and bimolecular fluorescence complementation. Secreted from the subventral pharyngeal glands into giant cells, Mj-NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity. The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of this process.

Comparative proteomics analysis of samples from hepatic cystic echinococcosis patients using data-independent acquisition approach.

Cystic echinococcosis is a zoonotic disease resulting from infection caused by the larval stage of Echinococcus granulosus. This study aimed to assess the specific proteins that are potential candidates for the development of a vaccine against E. granulosus. The data-independent acquisition approach was employed to identify differentially expressed proteins (DEPs) in E. granulosus samples. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was employed to identify several noteworthy proteins. Results: The DEPs in E. granulosus samples were identified (245 pericystic wall vs. parasite-free yellowish granuloma (PYG, 1725 PY vs. PYG, 2274 PN vs. PYG). Further examination of these distinct proteins revealed their predominant enrichment in metabolic pathways, amyotrophic lateral sclerosis, and neurodegeneration-associated pathways. Notably, among these DEPs, SH3BGRL, MST1, TAGLN2, FABP5, UBE2V2, and RARRES2 exhibited significantly higher expression levels in the PYG group compared with the PY group (P < 0.05). The findings may contribute to the understanding of the pathological mechanisms underlying echinococcosis, providing valuable insights into the development of more effective diagnostic tools, treatment modalities, and preventive strategies. SIGNIFICANCE: CE is a major public health hazard in the western regions of China, Central Asia, South America, the Mediterranean countries, and eastern Africa. Echinococcus granulosus is responsible for zoonotic disease through infection Our analysis focuses on the proteins in various samples by data-dependent acquisition (DIA) for proteomic analysis. The importance of this research is to develop new strategies and targets to protect against E. granulosus infections in humans.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis.

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Sja-let-7 suppresses the development of liver fibrosis via Schistosoma japonicum extracellular vesicles.

Schistosomiasis is a fatal zoonotic parasitic disease that also threatens human health. The main pathological features of schistosomiasis are granulomatous inflammation and subsequent liver fibrosis, which is a complex, chronic, and progressive disease. Extracellular vesicles (EVs) derived from schistosome eggs are broadly involved in host-parasite communication and act as important contributors to schistosome-induced liver fibrosis. However, it remains unclear whether substances secreted by the EVs of Schistosoma japonicum, a long-term parasitic "partner" in the hepatic portal vein of the host, also participate in liver fibrosis. Here, we report that EVs derived from S. japonicum worms attenuated liver fibrosis by delivering sja-let-7 into hepatic stellate cells (HSCs). Mechanistically, activation of HSCs was reduced by targeting collagen type I alpha 2 chain (Col1α2) and downregulation of the TGF-ß/Smad signaling pathway both in vivo and in vitro. Overall, these results contribute to further understanding of the molecular mechanisms underlying host-parasite interactions and identified the sja-let-7/Col1α2/TGF-ß/Smad axis as a potential target for treatment of schistosomiasis-related liver fibrosis.

Serum IgA contributes to the comprehension of Anisakis simplex associated chronic urticaria.

The phenotype of allergic diseases associated with Anisakis determines the pattern of cytokines related to antibody production. However, the role of serum IgA and the immunomodulatory mechanisms exerted by active infection of L3 or passive mucosal contact with A. simplex specific antigens has not been studied before. We measured serum cytokine by flow cytometry (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, TGF-ß1) and antibody levels (IgE, IgG4, IgA) by ELISA against total and excretory-secretory (ES) antigens, Ani s 3,and the group of major allergens Ani s 1, Ani s 7, and Ani s 13 in sera from 10 patients with gastro-allergic anisakiasis (GAA), 11 Anisakis sensitization associated chronic urticaria (CU+) as well as 17 non-Anisakis-sensitized patients with chronic urticaria (CU-), compared with the urticaria control group (18 subjects). Specific IgE, IgG4 and IgA were high in the GAA, but IgA levels were significantly higher in the CU+ with respect the CONTROL group. We observed higher levels of the ratio IgA/IgG4 in CU+ than GAA group for Ani s 1, Ani s 7, Ani s 13 and ES. Furthermore, chronic urticaria (CU) patients showed significant lower levels of IL-10, IFN-γ and IL-17A than patients without CU. The anti-Ani s 13 IgA/IgG4 ratio correlated positively with pro-inflammatory cytokines and ratios (TNF-α, IL-17A, Th17/Th2, Type1/Type2 and TNF-α/IL-10) in CONTROL group. In general, Anti-Anisakis IgA/G4 ratio was high in CU patients. In conclusion, this study demonstrates the importance of serum IgA because it is associated with chronic urticaria independently of Anisakis sensitization.

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.

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.

Trichinella spiralis cathepsin B bound and degraded host's intestinal type I collagen.

Trichinellosis is a zoonotic parasitic disease that poses threats to human health, the meat industry, food safety, and huge financial losses. The critical stage of Trichinella spiralis (T. spiralis) infection is the invasion of intestinal larvae into the host's intestinal epithelial cells (IECs). T. spiralis Cathepsin B (TsCB) specifically interacts with IECs to facilitate the invasion of larvae. This study aims to look at how TsCB affects mouse IECs. TsCB was successfully cloned, expressed, and characterized, demonstrating its natural cysteine protease hydrolysis activity. A total of 140 proteins that interact with rTsCB were identified by GST pull-down combined with LC-MS/MS, including type I collagen, an essential component of the host's intestinal epithelial barrier system and intimately related to intestinal epithelial damage. TsCB transcription and expression levels rise, whereas type I collagen in the host's intestinal mucosa declines when the T. spiralis larvae invaded. Besides, it was discovered that TsCB bound to and degraded type I collagen of the host's intestine. This research can serve as a foundation for clarifying how T. spiralis invades the host's intestinal barrier and might provide information on potential targets for the creation of novel treatments to treat parasite illnesses.

Characterization of a novel dipeptidyl peptidase 1 of Trichinella spiralis and its participation in larval invasion.

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines.

Metabolite profiling of Trichinella spiralis adult worms and muscle larvae identifies their excretory and secretory products.

Human trichinellosis is a parasitic infection caused by roundworms belonging to the genus Trichinella, especially Trichinella spiralis. Early and accurate clinical diagnoses of trichinellosis are required for efficacious prognosis and treatment. Current drug therapies are limited by antiparasitic resistance, poor absorption, and an inability to kill the encapsulating muscle-stage larvae. Therefore, reliable biomarkers and drug targets for novel diagnostic approaches and anthelmintic drugs are required. In this study, metabolite profiles of T. spiralis adult worms and muscle larvae were obtained using mass spectrometry-based metabolomics. In addition, metabolite-based biomarkers of T. spiralis excretory-secretory products and their related metabolic pathways were characterized. The metabolic profiling identified major, related metabolic pathways involving adenosine monophosphate (AMP)-dependent synthetase/ligase and glycolysis/gluconeogenesis in T. spiralis adult worms and muscle larvae, respectively. These pathways are potential drug targets for the treatment of the intestinal and muscular phases of infection. The metabolome of larva excretory-secretory products was characterized, with amino acid permease and carbohydrate kinase being identified as key metabolic pathways. Among six metabolites, decanoyl-l-carnitine and 2,3-dinor-6-keto prostaglandin F1α-d9 were identified as potential metabolite-based biomarkers that might be related to the host inflammatory processes. In summary, this study compared the relationships between the metabolic profiles of two T. spiralis growth stages. Importantly, the main metabolites and metabolic pathways identified may aid the development of novel clinical diagnostics and therapeutics for human trichinellosis and other related helminthic infections.

Excretory/secretory products from Trichinella spiralis adult worms ameliorate myocardial infarction by inducing M2 macrophage polarization in a mouse model.

BACKGROUND: Ischemia-induced inflammatory response is the main pathological mechanism of myocardial infarction (MI)-caused heart tissue injury. It has been known that helminths and worm-derived proteins are capable of modulating host immune response to suppress excessive inflammation as a survival strategy. Excretory/secretory products from Trichinella spiralis adult worms (Ts-AES) have been shown to ameliorate inflammation-related diseases. In this study, Ts-AES were used to treat mice with MI to determine its therapeutic effect on reducing MI-induced heart inflammation and the immunological mechanism involved in the treatment. METHODS: The MI model was established by the ligation of the left anterior descending coronary artery, followed by the treatment of Ts-AES by intraperitoneal injection. The therapeutic effect of Ts-AES on MI was evaluated by measuring the heart/body weight ratio, cardiac systolic and diastolic functions, histopathological change in affected heart tissue and observing the 28-day survival rate. The effect of Ts-AES on mouse macrophage polarization was determined by stimulating mouse bone marrow macrophages in vitro with Ts-AES, and the macrophage phenotype was determined by flow cytometry. The protective effect of Ts-AES-regulated macrophage polarization on hypoxic cardiomyocytes was determined by in vitro co-culturing Ts-AES-induced mouse bone marrow macrophages with hypoxic cardiomyocytes and cardiomyocyte apoptosis determined by flow cytometry. RESULTS: We observed that treatment with Ts-AES significantly improved cardiac function and ventricular remodeling, reduced pathological damage and mortality in mice with MI, associated with decreased pro-inflammatory cytokine levels, increased regulatory cytokine expression and promoted macrophage polarization from M1 to M2 type in MI mice. Ts-AES-induced M2 macrophage polarization also reduced apoptosis of hypoxic cardiomyocytes in vitro. CONCLUSIONS: Our results demonstrate that Ts-AES ameliorates MI in mice by promoting the polarization of macrophages toward the M2 type. Ts-AES is a potential pharmaceutical agent for the treatment of MI and other inflammation-related diseases.

Molecular characterization and functional implications on mouse peripheral blood mononuclear cells of annexin proteins from Echinococcus granulosus sensu lato.

BACKGROUND: Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE. METHODS: Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs). RESULTS: In this study, we identified four EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production. CONCLUSIONS: Our findings suggested that among the four identified EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells.

In vitro knockdown of TsDNase II-7 suppresses Trichinella spiralis invasion into the host's intestinal epithelial cells.

Trichinella spiralis (T. spiralis) adult-specific deoxyribonuclease II-7 (TsDNase II-7), a member of the DNase II-like nuclease family with no DNase II activity, was identified in the excretory-secretory (ES) products of adult worms (AWs). However, its biological functions are still unclear. Our previous study revealed that TsDNase II-7 is located around the infection site in the intestinal tissue, speculating that it was involved in the T. spiralis invasion of host intestinal epithelial cells (IECs). This study aimed to use RNA interference to verify our speculation that TsDNase II-7 in 3-day old adult T. spiralis (Ad3) plays a role in intestinal invasion. TsDNase II-7-specific small interfering RNAs (siRNAs) were delivered into muscle larvae (MLs) to knockdown TsDNase II-7 expression by electroporation. Twenty-four hours later, the MLs transfected with 2 µM siRNA-841 exhibited decreased in TsDNase II-7 transcription and expression as compared to the control MLs. The knockdown of TsDNase II-7 expression did not affect ML viability, and the low expression of TsDNase II-7 still maintained in Ad3 recovered from TsDNase II-7-RNAi-ML infected mice, resulting in a weakened ability of Ad3 to invade intestinal epithelial cells (IECs). These results indicated that knockdown of TsDNase II-7 gene expression via RNA interference (RNAi) suppressed adult worm invasion and confirmed that TsDNase II-7 plays a crucial role during the intestinal phase of T. spiralis infections, which provided new candidate for vaccine development of T. spiralis.

In silico secretome analyses of the polyphagous root-knot nematode Meloidogyne javanica: a resource for studying M. javanica secreted proteins.

BACKGROUND: Plant-parasitic nematodes (PPNs) that cause most damage include root-knot nematodes (RKNs) which are a major impediment to crop production. Root-knot nematodes, like other parasites, secrete proteins which are required for parasite proliferation and survival within the host during the infection process. RESULTS: Here, we used various computational tools to predict and identify classically and non-classically secreted proteins encoded in the Meloidogyne javanica genome. Furthermore, functional annotation analysis was performed using various integrated bioinformatic tools to determine the biological significance of the predicted secretome. In total, 7,458 proteins were identified as secreted ones. A large percentage of this secretome is comprised of small proteins of ≤ 300 aa sequence length. Functional analyses showed that M. javanica secretome comprises cell wall degrading enzymes for facilitating nematode invasion, and migration by disintegrating the complex plant cell wall components. In addition, peptidases and peptidase inhibitors are an important category of M. javanica secretome involved in compatible host-nematode interactions. CONCLUSION: This study identifies the putative secretome encoded in the M. javanica genome. Future experimental validation analyses can greatly benefit from this global analysis of M. javanica secretome. Equally, our analyses will advance knowledge of the interaction between plants and nematodes.

The helminth derived peptide FhHDM-1 redirects macrophage metabolism towards glutaminolysis to regulate the pro-inflammatory response.

We have previously identified an immune modulating peptide, termed FhHDM-1, within the secretions of the liver fluke, Fasciola hepatica, which is sufficiently potent to prevent the progression of type 1 diabetes and multiple sclerosis in murine models of disease. Here, we have determined that the FhHDM-1 peptide regulates inflammation by reprogramming macrophage metabolism. Specifically, FhHDM-1 switched macrophage metabolism to a dependence on oxidative phosphorylation fuelled by fatty acids and supported by the induction of glutaminolysis. The catabolism of glutamine also resulted in an accumulation of alpha ketoglutarate (α-KG). These changes in metabolic activity were associated with a concomitant reduction in glycolytic flux, and the subsequent decrease in TNF and IL-6 production at the protein level. Interestingly, FhHDM-1 treated macrophages did not express the characteristic genes of an M2 phenotype, thereby indicating the specific regulation of inflammation, as opposed to the induction of an anti-inflammatory phenotype per se. Use of an inactive derivative of FhHDM-1, which did not modulate macrophage responses, revealed that the regulation of immune responses was dependent on the ability of FhHDM-1 to modulate lysosomal pH. These results identify a novel functional association between the lysosome and mitochondrial metabolism in macrophages, and further highlight the significant therapeutic potential of FhHDM-1 to prevent inflammation.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.