MIG-23 is involved in sperm migration by modulating extracellular ATP levels in Ascaris suum.

In nematodes, spermiogenesis is a process of sperm activation in which nonmotile spermatids are transformed into crawling spermatozoa. Sperm motility acquisition during this process is essential for successful fertilization, but the underlying mechanisms remain to be clarified. Herein, we have found that extracellular adenosine-5'-triphosphate (ATP) level regulation by MIG-23, which is a homolog of human ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), was required for major sperm protein (MSP) filament dynamics and sperm motility in the nematode Ascaris suum. During sperm activation, a large amount of ATP was produced in mitochondria and was stored in refringent granules (RGs). Some of the produced ATP was released to the extracellular space through innexin channels. MIG-23 was localized in the sperm plasma membrane and contributed to the ecto-ATPase activity of spermatozoa. Blocking MIG-23 activity resulted in a decrease in the ATP hydrolysis activity of spermatozoa and an increase in the depolymerization rate of MSP filaments in pseudopodia, which eventually affected sperm migration. Overall, our data suggest that MIG-23, which contributes to the ecto-ATPase activity of spermatozoa, regulates sperm migration by modulating extracellular ATP levels.

Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum.

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.

Potential modulating effect of the Ascaris suum nicotinic acetylcholine receptor (nAChR) by compounds GSK575594A, diazepam and flumazenil discovered by structure-based virtual screening approach.

Parasitic infections are a widespread health problem and research of novel anthelmintic compounds is of the utmost importance. In this study we performed a virtual screening campaign by coupling ligand-based pharmacophore, homology modeling and molecular docking. The virtual screening campaign was conducted using a joined pool of the Drugbank database and a library of purchasable compounds in order to identify drug like compounds with similar pharmacological activity. Our aim was to identify compounds with a potential antihelmintic modulatory effect on nicotinic acetylcholine receptors (nAChR). We derived a 3D pharmacophore model based on the chemical features of known Ascaris suum nAChR modulators. To evaluate the in silico predictions, we tested selected hit compounds in contraction assays using somatic muscle flaps of the Ascaris suum neuromuscular tissue. We tested the modulatory effects of GSK575594A, diazepam and flumazenil hit compounds on nematode contractions induced by acetyl choline (ACh). The compound GSK575594A (3 µM) increased the Emax by 21 % with the EC50 dose ratio of 0.96. Diazepam (100 µM) decreased the Emax by 15 % (1.11 g to 0.95 g) with the EC50 ratio of 1.42 (shifted to the left from 11.25 to 7.93). Flumazenil decreased the EC50 value (from 11.22 µM to 4.88 µM) value showing dose ratio of 2.30, and increased the Emax by 4 % (from 1.54 g to 1.59 g). The observed biological activity was rationalized by molecular docking calculations. Docking scores were calculated against several binding sites within the Ascaris suum homology model. We constructed the homology model using the ACR-16 subunit sequence. The compound GSK575594A showed strong affinity for the intersubunit allosteric binding site within the nAChR transmembrane domain. The binding modes of diazepam and flumazenil suggest that these compounds have a comparable affinity for orthosteric and allosteric nAChR binding sites. The selected hit compounds displayed potential for further optimization as lead compounds. Therefore, such compounds may be useful in neutralizing the growing resistance of parasites to drugs, either alone or in combination with existing conventional anthelmintics.

Cholinergic receptors on intestine cells of Ascaris suum and activation of nAChRs by levamisole.

Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B).

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor.

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

Protective immunity elicited by the nematode-conserved As37 recombinant protein against Ascaris suum infection.

BACKGROUND: Ascaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: As37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 µg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules. CONCLUSIONS/SIGNIFICANCE: As37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections.

Body fluid from the parasitic worm Ascaris suum inhibits broad-acting pro-inflammatory programs in dendritic cells.

Dendritic cells (DCs) are essential for generating T-cell-based immune responses through sensing of potential inflammatory and metabolic cues in the local environment. However, there is still limited insight into the processes defining the resultant DC phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Here we compared the ability of a selected number of molecules to modulate the pro-inflammatory phenotype of lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated human monocyte-derived DCs towards an anti-inflammatory or regulatory phenotype, including Ascaris suum body fluid [helminth pseudocoelomic fluid (PCF)], the metabolites succinate and butyrate, and the type 2 cytokines thymic stromal lymphopoietin and interleukin-25. Our data show that helminth PCF and butyrate treatment suppress the T helper type 1 (Th1)-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS + IFN-γ-matured DCs by down-regulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in Toll-like receptor 4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, and transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were up-regulated. Collectively, our results further understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode.

Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten ß-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst ß-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes.

Galectin-2 suppresses nematode development by binding to the invertebrate-specific galactoseß1-4fucose glyco-epitope.

Galactoseß1-4Fucose (GalFuc) is a unique disaccharide found in invertebrates including nematodes. A fungal galectin CGL2 suppresses nematode development by recognizing the galactoseß1-4fucose epitope. The Caenorhabditis elegans galectin LEC-6 recognizes it as an endogenous ligand and the Glu67 residue of LEC-6 is responsible for this interaction. We found that mammalian galectin-2 (Gal-2) also has a comparable glutamate residue, Glu52. In the present study, we investigated the potential nematode-suppressing activity of Gal-2 using C. elegans as a model and focusing on Gal-2 binding to the GalFuc epitope. Gal-2 suppressed C. elegans development whereas its E52D mutant (Glu52 substituted by Asp), galectin-1 and galectin-3 had little effect on C. elegans growth. Lectin-staining using fluorescently-labeled Gal-2 revealed that, like CGL2, it specifically binds to the C. elegans intestine. Natural C. elegans glycoconjugates were specifically bound by immobilized Gal-2. Western blotting with anti-GalFuc antibody showed that the bound glycoconjugates had the GalFuc epitope. Frontal affinity chromatography with pyridylamine-labeled C. elegans N-glycans disclosed that Gal-2 (but not its E52D mutant) recognizes the GalFuc epitope. Gal-2 also binds to the GalFuc-bearing glycoconjugates of Ascaris and the GalFuc epitope is present in the parasitic nematodes Nippostrongylus brasiliensis and Brugia pahangi. These results indicate that Gal-2 suppresses C. elegans development by binding to its GalFuc epitope. The findings also imply that Gal-2 may prevent infestations of various parasitic nematodes bearing the GalFuc epitope.

Oxfendazole kinetics in pigs: In vivo assessment of its pattern of accumulation in Ascaris suum.

Ascaris suum is a widespread parasitic nematode that causes infection in pigs with high prevalence rates. Oxfendazole (OFZ) is effective against A. suum when used at a single high oral dose of 30 mg/kg. The aim of this study was to assess the pattern of distribution/accumulation of OFZ and its metabolites, in bloodstream (plasma), mucosal tissue and contents from small and large intestine and adult specimens of A. suum collected from infected and treated pigs. The activity of glutathione-S-transferases (GSTs) in A. suum was also investigated. Infected pigs were orally treated with OFZ (30 mg/kg) and sacrificed at 0, 3, 6 and 12 h after treatment. Samples of blood, mucosa and contents from both small and large intestine as well as adult worms were obtained and processed for quantification of OFZ/metabolites by HPLC. OFZ was the main analyte measured in all of the evaluated matrixes. The highest drug concentrations were determined in small (AUC0-t 718.7 ±â€¯283.5 µg h/g) and large (399.6 ±â€¯110.5 µg h/g) intestinal content. Concentrations ranging from 1.35 to 2.60 µg/g (OFZ) were measured in adult A. suum. GSTs activity was higher after exposure to OFZ both in vivo and ex vivo. The data obtained here suggest that the pattern of OFZ accumulation in A. suum would be more related to the concentration achieved in the fluid and mucosa of the small intestine than in other tissues/fluids. It is expected that increments in the amount of drug attained in the tissues/fluids of parasite location will correlate with increased drug concentration within the target parasite, and therefore with the resultant treatment efficacy. The results are particularly relevant considering the potential of OFZ to be used for soil transmitted helminths (STH) control programs and the advantages of pigs as a model to assess drug treatment to be implemented in humans.

The Intestinal Roundworm Ascaris suum Releases Antimicrobial Factors Which Interfere With Bacterial Growth and Biofilm Formation.

Ascariasis is a widespread soil-transmitted helminth infection caused by the intestinal roundworm Ascaris lumbricoides in humans, and the closely related Ascaris suum in pigs. Progress has been made in understanding interactions between helminths and host immune cells, but less is known concerning the interactions of parasitic nematodes and the host microbiota. As the host microbiota represents the direct environment for intestinal helminths and thus a considerable challenge, we studied nematode products, including excretory-secretory products (ESP) and body fluid (BF), of A. suum to determine their antimicrobial activities. Antimicrobial activities against gram-positive and gram-negative bacterial strains were assessed by the radial diffusion assay, while effects on biofilm formation were assessed using the crystal violet static biofilm and macrocolony assays. In addition, bacterial neutralizing activity was studied by an agglutination assay. ESP from different A. suum life stages (in vitro-hatched L3, lung-stage L3, L4, and adult) as well as BF from adult males were analyzed by mass spectrometry. Several proteins and peptides with known and predicted roles in nematode immune defense were detected in ESP and BF samples, including members of A. suum antibacterial factors (ASABF) and cecropin antimicrobial peptide families, glycosyl hydrolase enzymes such as lysozyme, as well as c-type lectin domain-containing proteins. Native, unconcentrated nematode products from intestine-dwelling L4-stage larvae and adults displayed broad-spectrum antibacterial activity. Additionally, adult A. suum ESP interfered with biofilm formation by Escherichia coli, and caused bacterial agglutination. These results indicate that A. suum uses a variety of factors with broad-spectrum antibacterial activity to affirm itself within its microbe-rich environment in the gut.

Comparative analysis of codon usage pattern and its influencing factors in Schistosoma japonicum and Ascaris suum.

Schistosoma japonicum and Ascaris suum are considered as the major parasites of human which cause various life threatening diseases such as schistomiasis and ascariasis. The codon usage bias (CUB) is known as the phenomenon of more usage of a specific codon than the other synonymous codons for an amino acid. The factors that influence the codon usage bias are mutation pressure, natural selection, gene expression, gene length, GC content, RNA stability, recombination rates, codon position etc. Here we had used various bioinformatic tools and statistical analyses to understand the compositional features, expression level and codon usage bias in the genes of these two species.After estimating the effective number of codon (ENC) in both the species, codon usage bias was found to be low and gene expression was high. The nucleobase A and T were used most often than C and G. From neutrality plot and correspondence analysis it was found that both natural selection and mutation pressure played an important role in shaping the codon usage pattern of both species. Moreover, natural selection played a major role while mutation pressure played a minor role in shaping the codon usage bias in S. japonicum and A.suum. This is the first report on the codon usage biology in S. japonicum and A.suum, and the factors influencing their codon usage bias. These results are expected to be useful for genetic engineering and evolutionary studies.

(S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu-ACR-16 receptor.

Nematode parasites infect ∼2 billion people world-wide. Infections are treated and prevented by anthelmintic drugs, some of which act on nicotinic acetylcholine receptors (nAChRs). There is an unmet need for novel therapeutic agents because of concerns about the development of resistance. We have selected Asu-ACR-16 from a significant nematode parasite genus, Ascaris suum, as a pharmaceutical target and nicotine as our basic moiety (EC50 6.21 ± 0.56 µM, Imax 82.39 ± 2.52%) to facilitate the development of more effective anthelmintics. We expressed Asu-ACR-16 in Xenopus oocytes and used two-electrode voltage clamp electrophysiology to determine agonist concentration-current-response relationships and determine the potencies (EC50s) of the agonists. Here, we describe the synthesis of a novel agonist, (S)-5-ethynyl-anabasine, and show that it is more potent (EC50 0.14 ± 0.01 µM) than other nicotine alkaloids on Asu-ACR-16. Agonists acting on ACR-16 receptors have the potential to circumvent drug resistance to anthelmintics, like levamisole, that do not act on the ACR-16 receptors.

Differential Chromosomal Localization of Centromeric Histone CENP-A Contributes to Nematode Programmed DNA Elimination.

The stability of the genome is paramount to organisms. However, diverse eukaryotes carry out programmed DNA elimination in which portions or entire chromsomes are lost in early development or during sex determination. During early development of the parasitic nematode, Ascaris suum, 13% of the genome is eliminated. How different genomic segments are reproducibly retained or discarded is unknown. Here, we show that centromeric histone CENP-A localization plays a key role in this process. We show that Ascaris chromosomes are holocentric during germline mitoses, with CENP-A distributed along their length. Prior to DNA elimination in the four-cell embryo, CENP-A is significantly diminished in chromosome regions that will be lost. This leads to the absence of kinetochores and microtubule attachment sites necessary for chromosome segregation, resulting in loss of these regions upon mitosis. Our data suggest that changes in CENP-A localization specify which portions of chromosomes will be lost during programmed DNA elimination.

Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues.

BACKGROUND AND PURPOSE: Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. EXPERIMENTAL APPROACH: Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. KEY RESULTS: Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. CONCLUSIONS AND IMPLICATIONS: We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.

The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.

Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 µM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 µM; fmoc-1, IC50, 10.00 ± 1.38 µM; fmoc-2, IC50, 16.67 ± 1.95 µM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.

Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

Effect of different N7 substitution of dinucleotide cap analogs on the hydrolytic susceptibility towards scavenger decapping enzymes (DcpS).

Scavenger decapping enzymes (DcpS) are involved in eukaryotic mRNA degradation process. They catalyze the cleavage of residual cap structure m(7)GpppN and/or short capped oligonucleotides resulting from exosom-mediated the 3' to 5' digestion. For the specific cap recognition and efficient degradation by DcpS, the positive charge at N7 position of guanine moiety is required. Here we examine the role the N7 substitution within the cap structure on the interactions with DcpS (human, Caenorhabditis elegans and Ascaris suum) comparing the hydrolysis rates of dinucleotide cap analogs (m(7)GpppG, et(7)GpppG, but(7)GpppG, bn(7)GpppG) and the binding affinities of hydrolysis products (m(7)GMP, et(7)GMP, but(7)GMP, bn(7)GMP). Our results show the conformational flexibility of the region within DcpS cap-binding pocket involved in the interaction with N7 substituted guanine, which enables accommodation of substrates with differently sized N7 substituents.

Different neuropeptides are expressed in different functional subsets of cholinergic excitatory motorneurons in the nematode Ascaris suum.

Neuropeptides are known to have dramatic effects on neurons and synapses; however, despite extensive studies of the motorneurons in the parasitic nematode Ascaris suum, their peptide content had not yet been described. We determined the peptide content of single excitatory motorneurons by mass spectrometry and tandem mass spectrometry. There are two subsets of ventral cord excitatory motorneurons, each with neuromuscular output either anterior or posterior to their cell body, mediating forward or backward locomotion, respectively. Strikingly, the two sets of neurons contain different neuropeptides, with AF9 and six novel peptides (As-NLP-21.1-6) in anterior projectors, and the six afp-1 peptides in addition to AF2 in posterior projectors. In situ hybridization confirmed the expression of these peptides, validating the integrity of the dissection technique. This work identifies new components of the functional behavioral circuit, as well as potential targets for antiparasitic drug development.

A globin domain in a neuronal transmembrane receptor of Caenorhabditis elegans and Ascaris suum: molecular modeling and functional properties.

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.