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Plants produce reactive oxygen species (ROS) upon infection, which typically trigger defence mechanisms and impede pathogen proliferation. Root-knot nematodes (RKNs, Meloidogyne spp.) represent highly detrimental pathogens capable of parasitizing a broad spectrum of crops, resulting in substantial annual agricultural losses. The involvement of ROS in RKN parasitism is well acknowledged. In this study, we identified a novel effector from Meloidogyne incognita, named CATLe, that contains a conserved catalase domain, exhibiting potential functions in regulating host ROS levels. Phylogenetic analysis revealed that CATLe is conserved across RKNs. Temporal and spatial expression assays showed that the CATLe gene was specifically up-regulated at the early infection stages and accumulated in the subventral oesophageal gland cells of M. incognita. Immunolocalization demonstrated that CATLe was secreted into the giant cells of the host plant during M. incognita parasitism. Transient expression of CATLe significantly dampened the flg22-induced ROS production in Nicotiana benthamiana. In planta assays confirmed that M. incognita can exploit CATLe to manipulate host ROS levels by directly degrading H2O2. Additionally, interfering with expression of the CATLe gene through double-stranded RNA soaking and host-induced gene silencing significantly attenuated M. incognita parasitism, highlighting the important role of CATLe. Taken together, our results suggest that RKNs can directly degrade ROS products using a functional catalase, thereby manipulating host ROS levels and facilitating parasitism.

Fatty acid-and retinol-binding proteins (FARs) belong to a unique family of excreted/secreted proteins (ESPs) found exclusively in nematodes. Much of our understanding of these proteins, however, is limited to their in vitro binding characteristics toward various fatty acids and retinol and has provided little insight into their in vivo functions or mechanisms. Recent research, however, has shown that FARs elicit an immunomodulatory role in plant and animal model systems, likely by sequestering lipids involved in immune signaling. This alludes to the intricate relationship between parasitic nematode effectors and their hosts.

Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P

The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-mobile systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. However, in nematodes the mode of action of SPT and its effect on their development have not been studied so far. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids and fatty acid composition. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by SPT, a well-known systemic insecticide for sucking pest control.

Protein ADP-ribosylation is a reversible post-translational modification (PTM) process that plays fundamental roles in cell signaling. The covalent attachment of ADP ribose polymers is executed by PAR polymerases (PARP) and it is essential for chromatin organization, DNA repair, cell cycle, transcription, and replication, among other critical cellular events. The process of PARylation or polyADP-ribosylation is dynamic and takes place across many tissues undergoing renewal and repair, but the molecular mechanisms regulating this PTM remain mostly unknown. Here, we introduce the use of the planarian Schmidtea mediterranea as a tractable model to study PARylation in the complexity of the adult body that is under constant renewal and is capable of regenerating damaged tissues. We identified the evolutionary conservation of PARP signaling that is expressed in planarian stem cells and differentiated tissues. We also demonstrate that Smed-PARP-3 homolog is required for proper regeneration of tissues in the anterior region of the animal. Furthermore, our results demonstrate, Smed-PARP-3(RNAi) disrupts the timely location of injury-induced cell death near the anterior facing wounds and also affects the regeneration of the central nervous system. Our work reveals novel roles for PARylation in large-scale regeneration and provides a simplified platform to investigate PARP signaling in the complexity of the adult body.

BackgroundThe full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used.Principal findingsTranscripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity.Conclusions/significanceThis in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated.

Parasitic helminths release molecular effectors into their hosts and these effectors can directly damage host tissue and modulate host immunity. Excreted/secreted proteins (ESPs) are one category of parasite molecular effectors that are critical to their success within the host. However, most studies of nematode ESPs rely on in vitro stimulation or culture conditions to collect the ESPs, operating under the assumption that in vitro conditions mimic actual in vivo infection. This assumption is rarely if ever validated. Entomopathogenic nematodes (EPNs) are lethal parasites of insects that produce and release toxins into their insect hosts and are a powerful model parasite system. We compared transcriptional profiles of individual Steinernema feltiae nematodes at different time points of activation under in vitro and in vivo conditions and found that some but not all time points during in vitro parasite activation have similar transcriptional profiles with nematodes from in vivo infections. These findings highlight the importance of experimental validation of ESP collection conditions. Additionally, we found that a suite of genes in the neuropeptide pathway were downregulated as nematodes activated and infection progressed in vivo, suggesting that these genes are involved in host-seeking behavior and are less important during active infection. We then characterized the ESPs of activated S. feltiae infective juveniles (IJs) using mass spectrometry and identified 266 proteins that are released by these nematodes. In comparing these ESPs with those previously identified in activated S. carpocapsae IJs, we identified a core set of 52 proteins that are conserved and present in the ESPs of activated IJs of both species. These core venom proteins include both tissue-damaging and immune-modulating proteins, suggesting that the ESPs of these parasites include both a core set of effectors as well as a specialized set, more adapted to the particular hosts they infect.

BackgroundSerine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis.Methodology/principal findingsSmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting.Conclusions/significanceThe data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

Mechanisms underlying anteroposterior body axis differences during adult tissue maintenance and regeneration are poorly understood. Here, we identify that post-translational modifications through the SUMO (Small Ubiquitin-like Modifier) machinery are evolutionarily conserved in the Lophotrocozoan Schmidtea mediterranea. Disruption of SUMOylation in adult animals by RNA-interference of the only SUMO E2 conjugating enzyme Ubc9 leads to a systemic increase in DNA damage and a remarkable regional defect characterized by increased cell death and loss of the posterior half of the body. We identified that Ubc9 is mainly expressed in planarian stem cells (neoblasts) but it is also transcribed in differentiated cells including neurons. Regeneration in Ubc9(RNAi) animals is impaired and associated with low neoblast proliferation. We present evidence indicating that Ubc9-induced regional cell death is preceded by alterations in transcription and spatial expression of repressors and activators of the Hedgehog signaling pathway. Our results demonstrate that SUMOylation acts as a regional-specific cue to regulate cell fate during tissue renewal and regeneration.

To reveal impacts of sexual mode on genome content, we compared chromosome-scale assemblies of the outcrossing nematode Caenorhabditis nigoni to its self-fertile sibling species, C. briggsaeC. nigoni's genome resembles that of outcrossing relatives but encodes 31% more protein-coding genes than C. briggsaeC. nigoni genes lacking C. briggsae orthologs were disproportionately small and male-biased in expression. These include the male secreted short (mss) gene family, which encodes sperm surface glycoproteins conserved only in outcrossing species. Sperm from mss-null males of outcrossing C. remanei failed to compete with wild-type sperm, despite normal fertility in noncompetitive mating. Restoring mss to C. briggsae males was sufficient to enhance sperm competitiveness. Thus, sex has a pervasive influence on genome content that can be used to identify sperm competition factors.

Organisms engage in extensive cross-species molecular dialog, yet the underlying molecular actors are known for only a few interactions. Many techniques have been designed to uncover genes involved in signaling between organisms. Typically, these focus on only one of the partners. We developed an expression quantitative trait locus (eQTL) mapping-based approach to identify cause-and-effect relationships between genes from two partners engaged in an interspecific interaction. We demonstrated the approach by assaying expression of 98 isogenic plants (Medicago truncatula), each inoculated with a genetically distinct line of the diploid parasitic nematode Meloidogyne hapla With this design, systematic differences in gene expression across host plants could be mapped to genetic polymorphisms of their infecting parasites. The effects of parasite genotypes on plant gene expression were often substantial, with up to 90-fold (P = 3.2 × 10-52) changes in expression levels caused by individual parasite loci. Mapped loci included a number of pleiotropic sites, including one 87-kb parasite locus that modulated expression of >60 host genes. The 213 host genes identified were substantially enriched for transcription factors. We distilled higher-order connections between polymorphisms and genes from both species via network inference. To replicate our results and test whether effects were conserved across a broader host range, we performed a confirmatory experiment using M. hapla-infected tomato. This revealed that homologous genes were similarly affected. Finally, to validate the broader utility of cross-species eQTL mapping, we applied the strategy to data from a Salmonella infection study, successfully identifying polymorphisms in the human genome affecting bacterial expression.

The plant-parasitic nematode Heterodera schachtii is an obligate biotroph that induces syncytial feeding sites in roots of its hosts. Nematodes produce effectors that are secreted into the host and facilitate infection process. Here we identified H. schachtii protein disulphide isomerase (HsPDI) as a putative effector that interferes with the host's redox status. In situ hybridization showed that HsPdi is specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2). HsPdi is up-regulated in the early parasitic J2s. Silencing of HsPdi by RNA interference in the J2s hampers their development and leads to structural malfunctions in associated feeding sites induced in Arabidopsis roots. Expression of HsPDI in Arabidopsis increases plant's susceptibility towards H. schachtii. HsPdi expression is up-regulated in the presence of exogenous H2O2, whereas HsPdi silencing results in increased mortality under H2O2 stress. Stable expression of HsPDI in Arabidopsis plants decreases ROS burst induced by flg22. Transiently expressed HsPDI in N. benthamiana leaves is localized in the apoplast. HsPDI plays an important role in the interaction between nematode and plant, probably through inducing local changes in the redox status of infected host tissue. It also contributes to protect the nematode from exogenous H2O2 stress.

The beet cyst nematode Heterodera schachtii causes major yield losses in sugar beet. Understanding the interaction between H. schachtii and its host plant is important for developing a sustainable management system. Nematode effectors play a crucial role in initializing and sustaining successful parasitism. In our study, we identified a gene (Hs-Tyr) encoding a tyrosinase functional domain (PF00264). We describe Hs-Tyr as a novel nematode effector. Hs-Tyr is localized in the nematode esophageal gland. Up-regulation of its expression coincided with the parasitic developmental stages of the nematode. Silencing Hs-Tyr by RNA interference made the treated nematodes less virulent. When RNAi-treated nematodes succeeded in infecting the plant, developing females and their associated syncytial nurse cells were significantly smaller than in control plants. Ectopically expressing the Hs-Tyr effector in Arabidopsis increased plant susceptibility to H. schachtii, but not to the root-knot nematode Meloidogyne incognita. Interestingly, Hs-Tyr in the plant promoted plant growth and changed the root architecture. Additionally, the expression of Hs-Tyr in Arabidopsis caused changes in the homeostasis of several plant hormones especially auxin and the ethylene precursor aminocyclopropane-carboxylic acid.

Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks.

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.

Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.

The X-ray crystal structure of arginase from Schistosoma mansoni (SmARG) and the structures of its complexes with several amino acid inhibitors have been determined at atomic resolution. SmARG is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea, and this enzyme is upregulated in all forms of the parasite that interact with the human host. Current hypotheses suggest that parasitic arginases could play a role in host immune evasion by depleting pools of substrate l-arginine that would otherwise be utilized for NO biosynthesis and NO-dependent processes in the immune response. Although the amino acid sequence of SmARG is only 42% identical with that of human arginase I, residues important for substrate binding and catalysis are strictly conserved. In general, classical amino acid inhibitors such as 2(S)-amino-6-boronohexanoic acid (ABH) tend to bind more weakly to SmARG than to human arginase I despite identical inhibitor binding modes in each enzyme active site. The identification of a patch on the enzyme surface capable of accommodating the additional Cα substitutent of an α,α-disubstituted amino acid inhibitor suggests that such inhibitors could exhibit higher affinity and biological activity. The structures of SmARG complexed with two different α,α-disubstituted derivatives of ABH are presented and provide a proof of concept for this approach in the enhancement of enzyme-inhibitor affinity.

Nematodes compose an abundant and diverse invertebrate phylum with members inhabiting nearly every ecological niche. Panagrellus redivivus (the "microworm") is a free-living nematode frequently used to understand the evolution of developmental and behavioral processes given its phylogenetic distance to Caenorhabditis elegans. Here we report the de novo sequencing of the genome, transcriptome, and small RNAs of P. redivivus. Using a combination of automated gene finders and RNA-seq data, we predict 24,249 genes and 32,676 transcripts. Small RNA analysis revealed 248 microRNA (miRNA) hairpins, of which 63 had orthologs in other species. Fourteen miRNA clusters containing 42 miRNA precursors were found. The RNA interference, dauer development, and programmed cell death pathways are largely conserved. Analysis of protein family domain abundance revealed that P. redivivus has experienced a striking expansion of BTB domain-containing proteins and an unprecedented expansion of the cullin scaffold family of proteins involved in multi-subunit ubiquitin ligases, suggesting proteolytic plasticity and/or tighter regulation of protein turnover. The eukaryotic release factor protein family has also been dramatically expanded and suggests an ongoing evolutionary arms race with viruses and transposons. The P. redivivus genome provides a resource to advance our understanding of nematode evolution and biology and to further elucidate the genomic architecture leading to free-living lineages, taking advantage of the many fascinating features of this worm revealed by comparative studies.

BackgroundThe barber's pole worm, Haemonchus contortus, is one of the most economically important parasites of small ruminants worldwide. Although this parasite can be controlled using anthelmintic drugs, resistance against most drugs in common use has become a widespread problem. We provide a draft of the genome and the transcriptomes of all key developmental stages of H. contortus to support biological and biotechnological research areas of this and related parasites.ResultsThe draft genome of H. contortus is 320 Mb in size and encodes 23,610 protein-coding genes. On a fundamental level, we elucidate transcriptional alterations taking place throughout the life cycle, characterize the parasite's gene silencing machinery, and explore molecules involved in development, reproduction, host-parasite interactions, immunity, and disease. The secretome of H. contortus is particularly rich in peptidases linked to blood-feeding activity and interactions with host tissues, and a diverse array of molecules is involved in complex immune responses. On an applied level, we predict drug targets and identify vaccine molecules.ConclusionsThe draft genome and developmental transcriptome of H. contortus provide a major resource to the scientific community for a wide range of genomic, genetic, proteomic, metabolomic, evolutionary, biological, ecological, and epidemiological investigations, and a solid foundation for biotechnological outcomes, including new anthelmintics, vaccines and diagnostic tests. This first draft genome of any strongylid nematode paves the way for a rapid acceleration in our understanding of a wide range of socioeconomically important parasites of one of the largest nematode orders.

Objectives: We compared the effect of 12 weeks lesser mealworm-based (Alphitobius diaperinus) protein supplementation to whey protein and placebo supplementation on Exercise-Induced Muscle Damage (EIMD) after long-distance walking in older adults., Methods: in this randomized controlled trial, 70 physically active older adults (>60 years) were randomly allocated to the following groups: I) lesser mealworm protein, II) whey protein or III) iso-caloric placebo. Participants received supplements 11 weeks before and 1 week during a 3-day long-distance walking challenge (30-50 km per day). Blood concentrations of creatinine kinase (CK) and lactate dehydrogenase (LDH), handgrip strength and muscle soreness were measured pre-exercise and directly after each walking bout., Results: Significant elevations of CK concentrations (103 [76-161] U/l to 758 [342-1104] U/l, p < 0.001) and LDH concentrations (202 [175-220] to 283 [252-339] U/l, p < 0.001) were observed following 7h45 min ± 11 min of walking exercise per day, but the magnitude of this effect did not differ among suppletion groups. Hand grip strength decreased significantly (p < 0.001) while muscle soreness increased (p = 0.002) after the first walking day compared to pre-exercise, with no group differences., Conclusion: 12-weeks of lesser mealworm-based protein supplementation (30 g/day) does not attenuate exercise induced muscle damage in older adults following three days of prolonged walking exercise in comparison to placebo or whey protein., (Copyright © 2024. Published by Elsevier Masson SAS.)

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-β1) 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., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

BackgroundThe possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.Methodology/principal findingsWe investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite.ConclusionsTransient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Schistosomiasis is a prevalent and chronic helmintic disease in tropical regions. Treatment and control relies on chemotherapy with just one drug, praziquantel and this reliance is of concern should clinically relevant drug resistance emerge and spread. Therefore, to identify potential target proteins for new avenues of drug discovery we have taken a comparative chemogenomics approach utilizing the putative proteome of Schistosoma mansoni compared to the proteomes of two model organisms, the nematode, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster. Using the genome comparison software Genlight, two separate in silico workflows were implemented to derive a set of parasite proteins for which gene disruption of the orthologs in both the model organisms yielded deleterious phenotypes (e.g., lethal, impairment of motility), i.e., are essential genes/proteins. Of the 67 and 68 sequences generated for each workflow, 63 were identical in both sets, leading to a final set of 72 parasite proteins. All but one of these were expressed in the relevant developmental stages of the parasite infecting humans. Subsequent in depth manual curation of the combined workflow output revealed 57 candidate proteins. Scrutiny of these for druggable protein homologs in the literature identified 35 S. mansoni sequences, 18 of which were homologous to proteins with 3D structures including co-crystallized ligands that will allow further structure-based drug design studies. The comparative chemogenomics strategy presented generates a tractable set of S. mansoni proteins for experimental validation as drug targets against this insidious human pathogen.

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.

BackgroundDuring invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.Methodology and resultsHuman skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in "triple play" mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.ConclusionsThis analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire.

The T-cell-mediated immune response exhibits a crucial function in the control of the intrahepatic proliferation of Echinococcus multilocularis larvae in mice and humans, both being natural intermediate hosts of the parasite. Antigen B (AgB), a metabolized Echinococcus spp. lipoprotein, contributes to the modulation of the T-cell immune response, and distinct sites of the corresponding AgB1, AgB3 and AgB4 genes were shown to be under positive selection pressure. Since several AgB gene variants are present in a single Echinococcus metacestode, we used secondary E. multilocularis infections in BALB/c and in athymic nude mice (devoid of T-cell responses) to analyze the effect of the cellular immune response on the expression and diversity of EmAgB1-EmAgB4 genes. We demonstrated hereby that EmAgB transcripts were less abundant in nude mice during the early phase of infection (at one month post-infection), and that EmAgB2 is simultaneously down-regulated when compared to the other three genes. A negative relationship exists between the level of transcription and diversity of EmAgB genes. Moreover, no excess of non-synonymous substitutions was found among the distinct EmAgB alleles from a single host. Together, these results pointed to the effect of purifying selection, which seemed to eliminate the detrimental AgB variants generated during the development of the metacestode within the peritoneal cavity of its intermediate host.

Numerous genetic variants of the Echinococcus antigen B (AgB) are encountered within a single metacestode. This could be a reflection of gene redundancy or the result of a somatic hypermutation process. We evaluate the complexity of the AgB multigene family by characterizing the upstream promoter regions of the 4 already known genes (EgAgB1-EgAgB4) and evaluating their redundancy in the genome of 3 Echinococcus species (E. granulosus, E. ortleppi and E. multilocularis) using PCR-based approaches. We have ascertained that the number of AgB gene copies is quite variable, both within and between species. The most repetitive gene seems to be AgB3, of which there are more than 110 copies in E. ortleppi. For E. granulosus, we have cloned and characterized 10 distinct upstream promoter regions of AgB3 from a single metacestode. Our sequences suggest that AgB1 and AgB3 are involved in gene conversion. These results are discussed in light of the role of gene redundancy and recombination in parasite evasion mechanisms of host immunity, which at present are known for protozoan organisms, but virtually unknown for multicellular parasites.

Twenty-seven PCR-derived antigen B (AgB) nucleotide sequences from four Echinococcus species (Echinococcus granulosus, Echinococcus multilocularis, Echinococcus oligarthrus and Echinococcus vogeli) were aligned with 78 already published sequences, to generate a maximum likelihood phylogeny of the AgB multigene family. The phylogenetic analysis confirms that the family is constituted by four groups of genes present in each one of the four species (AgB1, AgB2, AgB3 and AgB4), and suggests that it originated by ancient duplication events preceding speciation within the genus. AgB5 sequences, which had been formerly suggested to correspond to a putatively new AgB subunit, cluster with AgB3. Likelihood tests suggest that AgB gene evolution may have been driven by heterogeneous selection pressures acting on particular AgB1, AgB3 and AgB4 codons. No selection is detected in AgB2. We discuss implications of our findings in terms of AgB biology and its use as a diagnostic tool.

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.

Recent studies have demonstrated that the Echinococcus granulosus antigen B (AgB) interferes with the intermediate hosts' immune response and is encoded by a multigene family. The number of members within the family is still uncertain, but there are several evidences of a large genetic variability. The E. granulosus AgB genomic sequences available in nucleotide databases can be grouped into four clades, corresponding to genes EgAgB1, EgAgB2, EgAgB3 and EgAgB4. In the present study, we use PCR amplifications followed by cloning and sequencing to evaluate the genetic variability for AgB isoforms. Two pairs of primers were independently used for PCR amplification. Both PCR reactions from each of three isolated protoscolex (larvae) were cloned in a plasmid vector and the plasmid inserts of 30 colonies from each cloning experiment were sequenced. Using phylogenetic tools, the 113 EgAgB clones are classified as follows: 25 are related to EgAgB1, 24 to EgAgB2, 9 to EgAgB3 and 39 to EgAgB4. The remaining 16 clones form a separate cluster, which we name EgAgB5, more closely related to EgAgB3 than to any of the other genes. Within each gene group, a number of variant sequences occur, which differ from one another by one or few nucleotides. One EgAgB3 clone has a premature stop codon (pseudogene) and an EgAgB2 clone lacks the region corresponding to the intron. The overall variation cannot be explained by differences among the asexual protoscoleces, or by experimental artifacts. Using Echinococcuss AgB genes from other species/strains as outgroups, neutrality is rejected for EgAgB2, and balancing selection is detected for EgAgB5, which also seems to be involved in gene conversion. We suggest that EgAgB1-EgAgB5 represent a family of contingency genes, that is, genes that are variably expressed, so that some but not others are expressed in each individual parasite. Contingency genes are common in parasitic protozoa and other microparasites, but the EgAgB family is the first set identified in a multicellular parasite.

BackgroundThe correct folding and dimerization of tubulins, before their addition to the microtubular structure, needs a group of conserved proteins called cofactors A to E. The biochemical analysis of cofactors gave an insight to their general functions, however not much is known about the domain structure and detailed, molecular function of these proteins.ResultsCombining modelling and fold prediction tools, we present 3D models of all cofactors, including several previously unannotated domains of cofactors B-E. Apart from the new HEAT and Armadillo domains in cofactor D and an unusual spectrin-like domain in cofactor C, we have identified a new subfamily of ubiquitin-like domains in cofactors B and E. Together, these observations provide a reliable, molecular level model of cofactor complex.ConclusionDistant homology searches allowed the identification of unknown regions of cofactors as self-reliant domains and allow us to present a detailed hypothesis of how a cofactor complex performs its function.

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.

Macromolecular structures called kinetochores attach and move chromosomes within the spindle during chromosome segregation. Using electron microscopy, we identified a structure on the holocentric mitotic and meiotic chromosomes of Caenorhabditis elegans that resembles the mammalian kinetochore. This structure faces the poles on mitotic chromosomes but encircles meiotic chromosomes. Worm kinetochores require the evolutionarily conserved HIM-10 protein for their structure and function. HIM-10 localizes to the kinetochores and mediates attachment of chromosomes to the spindle. Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction. HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans. Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.

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., (© 2023. The Author(s).)

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 microm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope-based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at approximately 1.1 microm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end-directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.