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.

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.

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.

SoxB family genes delay regeneration and cause abnormal movement in Dugesia japonica.

SoxB subfamily is an important branch of Sox family and plays a key role in animal physiological process, but little is known about their function in planarian regeneration. This study aims to evaluate the function of DjSoxB family genes in intact and regenerating planarians Dugesia japonica. Here, we amplify the full-length cDNA of DjSoxB1 and DjSoxB2 in D. japonica by rapid amplification of the cDNA ends (RACE), detect the expression of DjSoxB family genes in planarian. The results show that DjSoxBs are expressed in parenchymal tissue and the hybridization signals partially disappear after irradiation indicates DjSoxB family genes are expressed in neoblasts. After the RNA interference (RNAi) of DjSoxB1, DjSoxB2 and DjSoxB3 separately, the numbers of proliferative cells are all reduced that causes planarians show slower growth of blastema in the early stage of regeneration, and nerves of planarians are affected that the movement speed of planarians decreases in varying degrees. Specially, planarians in the DjSoxB3 RNAi group show shrinkage and twisting. Overall, this study reveals that DjSoxB family genes play a role in cell proliferation during regeneration. They also play an important role in the maintenance of normal nerve function and nerve regeneration. These results provide directions for the functional study of SoxB family genes and provide an important foundation for planarian regeneration.

Temperature Effects on Expression Levels of hsp Genes in Eggs and Second-Stage Juveniles of Meloidogyne hapla Chitwood, 1949.

Meloidogyne hapla is one of the most important nematode pathogens. It is a sedentary, biotrophic parasite of plants that overwinters in the soil or in diseased roots. The development of M. hapla is temperature dependent. Numerous studies have been performed on the effect of temperature on the development of M. hapla, but only a few of them analyzed the heat shock protein (hsp) genes. The aim of the study was to perform expression profiling of eight hsp genes (Mh-hsp90, Mh-hsp1, Mh-hsp4, Mh-hsp6, Mh-hsp60, Mh-dnj19, Mh-hsp43, and Mh-hsp12.2) at two development stages of M. hapla, i.e., in eggs and second-stage juveniles (J2). The eggs and J2 were incubated under cold stress (5 °C), heat stress (35 °C, 40 °C), and non-stress (10 °C, 20 °C, and 30 °C) conditions. Expression profiling was performed by qPCR. It was demonstrated that only two genes, Mh-hsp60 and Mh-dnj19, have been upregulated by heat and cold stress at both development stages. Heat stress upregulated the expression of more hsp genes than cold stress did. The level of upregulation of most hsp genes was more marked in J2 than in eggs. The obtained results suggest that the Mh-hsp90 and Mh-hsp1 genes can be used as bioindicators of environmental impacts on nematodes of the Meloidogyne genus.

Changes in Internal Structure and Dynamics upon Binding Stabilise the Nematode Anticoagulant NAPc2.

Abnormal blood coagulation is a major health problem and natural anticoagulants from blood-feeding organisms have been investigated as novel therapeutics. NAPc2, a potent nematode-derived inhibitor of coagulation, has an unusual mode of action that requires coagulation factor Xa but does not inhibit it. Molecular dynamics simulations of NAPc2 and factor Xa were generated to better understand NAPc2. The simulations suggest that parts of NAPc2 become more rigid upon binding factor Xa and reveal that two highly conserved residues form an internal salt bridge that stabilises the bound conformation. Clotting time assays with mutants confirmed the utility of the salt bridge and suggested that it is a conserved mechanism for stabilising the bound conformation of secondary structure-poor protease inhibitors.

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.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

PURPOSE: Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. METHODS: After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. RESULTS: The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. CONCLUSION: The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

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.

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.

Trichinella spiralis-Secreted Products Promote Collagen Capsule Formation through TGF-ß1/Smad3 Pathway.

Trichinella spiralis (T. spiralis) muscle larvae colonize in the host's skeletal muscle cells, which are surrounded by collagen capsules. The mechanism underlying muscle stage larva-induced collagen capsule formation remains unknown. To clarify the mechanism, a T. spiralis muscular-infected mouse model was established by a single lateral tail vein injection with 20,000 T. spiralis newborn larvae (NBL). The infected mice were treated with or without SB525334 (TGF-ß1 receptor type I inhibitor). Diaphragms were obtained post-infection, and the expression levels of the TGF-ß1/Smad3 pathway-related genes and collagen genes (type IV and VI) were observed during the process of collagen capsule formation. The changes in myoblasts under stimulation of the excretory-secretory (ES) products of NBL with or without SB525334 were further investigated. Results showed that the expression levels of type IV collagen gene, type VI collagen gene, Tgfb1, and Smad3 were significantly increased in infected mice muscle cells. The expression levels of all the above genes were enhanced by the products of NBL in myoblast cells. These changes were reversed by co-treatment with SB525334 in vivo and in vitro. In conclusion, the TGF-ß1/Smad3 pathway can be activated by T. spiralis infection in muscle cells. The activated TGF-ß1/Smad3 pathway can stimulate the secretion of collagens by myocytes and plays a promoting role in the process of collagen capsule formation. The research has the limitation that the protein identification of the products of NBL has yet to be performed. Therefore, the specific components in the T. spiralis ES products that induce collagen synthesis should be further investigated.

Identification of antigens in the Trichinella spiralis extracellular vesicles for serological detection of early stage infection in swine.

BACKGROUND: Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis. METHODS: Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA. RESULTS: Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 µg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis. CONCLUSIONS: This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis.

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.

Filarial DAF-12 sense the host serum to resume iL3 development during infection.

Nematode parasites enter their definitive host at the developmentally arrested infectious larval stage (iL3), and the ligand-dependent nuclear receptor DAF-12 contributes to trigger their development to adulthood. Here, we characterized DAF-12 from the filarial nematodes Brugia malayi and Dirofilaria immitis and compared them with DAF-12 from the non-filarial nematodes Haemonchus contortus and Caenorhabditis elegans. Interestingly, Dim and BmaDAF-12 exhibit high sequence identity and share a striking higher sensitivity than Hco and CelDAF-12 to the natural ligands Δ4- and Δ7-dafachronic acids (DA). Moreover, sera from different mammalian species activated specifically Dim and BmaDAF-12 while the hormone-depleted sera failed to activate the filarial DAF-12. Accordingly, hormone-depleted serum delayed the commencement of development of D. immitis iL3 in vitro. Consistent with these observations, we show that spiking mouse charcoal stripped-serum with Δ4-DA at the concentration measured in normal mouse serum restores its capacity to activate DimDAF-12. This indicates that DA present in mammalian serum participate in filarial DAF-12 activation. Finally, analysis of publicly available RNA sequencing data from B. malayi showed that, at the time of infection, putative gene homologs of the DA synthesis pathways are coincidently downregulated. Altogether, our data suggest that filarial DAF-12 have evolved to specifically sense and survive in a host environment, which provides favorable conditions to quickly resume larval development. This work sheds new light on the regulation of filarial nematodes development while entering their definitive mammalian host and may open the route to novel therapies to treat filarial infections.

Comparative proteomics analysis of adult Haemonchus contortus isolates from Ovis ammon.

Haemonchus contortus is an important parasite that causes disease that seriously endangers ruminant animals cattle, sheep, goat, and camel. Here, we compared the proeomics analysis of three adult Haemonchus contortus isolates from mouflons (Ovis ammon). A total of 1,299 adult worm proteins were identified, and 461 proteins were quantified, of which 82 (108), 83 (97), and 97 (86) significantly upregulated (downregulated) differentially expressed proteins (DEPs) were detected among pairwise comparisons (1-vs.-3, 2-vs.-3, and 2-vs.-1). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatic analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function, and catabolism pathways. In addition, Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out to screen the DEPs. The main biological processes involved were nucleotide, nucleotide phosphate, ribonucleotide, purine-containing compound, purine ribonucleotide, single-organism, oxoacid, organic, carboxylic, oxoacid metabolic processes and single-organism catabolic processes. The majority of KEGG pathways were found to be related to metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, and microbial metabolism in diverse environments. Moreover, we also found differences in the expression of some important or novel regulatory proteases, such as serine hydroxymethyl transferase (SHMT), dihydrolipoyl dehydrogenase (DLD), and transket pyr domain-containing protein (TKPD). In summary, label-free proteomic analysis of adult H. contortus worms displayed significant differences in three different individual isolates, which helps to improve our understanding of the growth and metabolic mechanisms of H. contortus in different individuals and relative natural environments and provides novel drug targets for the treatment of parasitic diseases.

The mechanism of acid resistance by ornithine decarboxylase in Trichinella spiralis.

Trichinella spiralis is a zoonotic parasite with worldwide distribution that can seriously harm human health and animal husbandry. Ornithine decarboxylase is a component of the acid resistance (AR) system in Escherichia coli. The aim of this study was to investigate the role that T. spiralis ornithine decarboxylase (TsODC) plays in the acid resistance mechanism of T. spiralis. This study involved assessing the transcription and expression of TsODC in worms under acidic conditions. According to mRNA sequences published by NCBI and the results of molecular biology experiments, the complete TsODC sequence was cloned and expressed. rTsODC had good immunogenicity, and immunofluorescence analysis revealed that TsODC was principally localized on the surface tissues of the nematode, especially at the head and tail. qRT‒PCR and Western blotting analysis indicated that the relative expression levels of TsODC mRNA and protein were highest when cultured at pH 2.5 for 2 h. The muscle larvae (ML) of T. spiralis were treated with curcumin and rapamycin, as well as arginine and TsODC polyantisera. The expression levels of TsODC mRNA and protein were significantly increased by arginine and suppressed by curcumin and rapamycin. After reducing the amount of TsODC, the relative expression of TsODC mRNA and the survival rate of T. spiralis ML were both reduced when compared to these values in the phosphate-buffered saline (PBS) group. The results indicated that TsODC is a member of the T. spiralis AR system and different treatments on TsODC have different effects; thus, these treatments might be a new way to prevent T. spiralis infection.

Discovery of Novel Effector Protein Candidates Produced in the Dorsal Gland of Adult Female Root-Knot Nematodes.

Root-knot nematodes (RKN) (Meloidogyne spp.) represent one of the most damaging groups of plant-parasitic nematodes. They secrete effector proteins through a protrusible stylet to manipulate host cells for their benefit. Stylet-secreted effector proteins are produced within specialized secretory esophageal gland cells, one dorsal gland (DG) and two subventral glands (SvG), whose activity differ throughout the nematode life cycle. Previous gland transcriptomic profiling studies identified dozens of candidate RKN effectors but were focused on the juvenile stages of the nematode, when the SvGs are most active. We developed a new approach to enrich for the active DGs of M. incognita adult female RKN for RNA and protein extraction. Female heads were manually cut from the body, and a combination of sonication and vortexing was used to dislodge contents inside the heads. DG-enriched fractions were collected by filtering, using cell strainers. Comparative transcriptome profiling of pre-parasitic second-stage juveniles, female heads, and DG-enriched samples was conducted using RNA sequencing. Application of an established effector mining pipeline led to the identification of 83 candidate effector genes upregulated in DG-enriched samples of adult females that code for proteins with a predicted signal peptide but lack transmembrane domains or homology to proteins in the free-living nematode Caenorhabditis elegans. In situ hybridization resulted in the identification of 14 new DG-specific candidate effectors expressed in adult females. Taken together, we have identified novel candidate Meloidogyne effector genes that may have essential roles during later stages of parasitism. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.