Structure-based virtual screening of highly potent inhibitors of the nematode chitinase CeCht1.

Nematode chitinases play vital roles in various physiological processes, including egg hatching, larva moulting, and reproduction. Small-molecule inhibitors of nematode chitinases have potential applications for controlling nematode pests. On the basis of the crystal structure of CeCht1, a representative chitinase indispensable to the eggshell chitin degradation of the model nematode Caenorhabditis elegans, we have discovered a series of novel inhibitors bearing a (R)-3,4-diphenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one scaffold by hierarchical virtual screening. The crystal structures of CeCht1 complexed with two of these inhibitors clearly elucidated their interactions with the enzyme active site. Based on the inhibitory mechanism, several analogues with improved inhibitory activities were identified, among which the compound PP28 exhibited the most potent activity with a Ki value of 0.18 µM. This work provides the structural basis for the development of novel nematode chitinase inhibitors.

Speciation and adaptive evolution reshape antioxidant enzymatic system diversity across the phylum Nematoda.

BACKGROUND: Nematodes have evolved to survive in diverse ecological niches and can be a serious burden on agricultural economy, veterinary medicine, and public health. Antioxidant enzymes in parasitic nematodes play a critical role in defending against host oxidative stress. However, the features of the evolution of antioxidant enzymes in the phylum Nematoda remain elusive. RESULTS: Here, we systematically investigated the evolution and gene expression of antioxidant enzymes in the genomes of 59 nematodes and transcriptomes of 20 nematodes. Catalase has been independently lost in several orders, suggesting that it is unnecessary for some nematodes. Unlike in mammals, phospholipid hydroperoxide glutathione peroxidase is widely distributed in nematodes, among which it has evolved independently. We found that superoxide dismutase (SOD) has been present throughout nematode evolutionary process, and the extracellular isoform (SOD3) is diverged from the corresponding enzyme in mammals and has undergone duplication and differentiation in several nematodes. Moreover, the evolution of intracellular and extracellular SOD isoforms in filaria strongly indicates that extracellular SOD3 originated from intracellular SOD1 and underwent rapid evolution to form the diversity of extracellular SOD3. We identify a novel putative metal-independent extracellular SOD presenting independently in Steinernema and Strongyloididae lineage that featured a high expression level in Strongyloides larvae. Sequence divergence of SOD3 between parasitic nematodes and their closest free-living nematode, the specifically high expression in the parasitic female stage, and presence in excretory-secretory proteome of Strongyloides suggest that SOD3 may be related with parasitism. CONCLUSIONS: This study advances our understanding of the complex evolution of antioxidant enzymes across Nematoda and provides targets for controlling parasitic nematode diseases.

Alternative splicing of coq-2 controls the levels of rhodoquinone in animals.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that synthesize RQ. We show that in Caenorhabditis elegans COQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.

Nematode endosymbiont competition: Fortune favors the fittest.

Endosymbiotic bacteria that obligately associate with entomopathogenic nematodes as a complex are a unique model system to study competition. These nematodes seek an insect host and provide entry for their endosymbionts. Through their natural products, the endosymbionts nurture their nematodes by eliminating secondary infection, providing nutrients through bioconversion of the insect cadaver, and facilitating reproduction. On one hand, they cooperatively colonize the insect host and neutralize other opportunistic biotic threats. On the other hand, inside the insect cadaver as a fighting pit, they fiercely compete for the fittest partnership that will grant them the reproductive dominance. Here, we review the protective and nurturing nature of endosymbiotic bacteria for their nematodes and how their selective preference shapes the superior nematode-endosymbiont pairs as we know today.

Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus.

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.

Molecular basis of pathogenic parasitic infections: insights from parasite kinome.

Infectious diseases caused by numerous parasitic pathogens represent a global health conundrum. Several animal and plant pathogens are responsible for causing acute illness in humans and deadly plant infections. These pathogens have evolved a diverse array of infection strategies and survival methods within the host organism. Recent research has highlighted the role of protein kinases in the overall virulence and pathogenicity of the pathogens. Protein kinases (Pks) are a group of enzymes known to catalyse the phosphorylation of a wide variety of cellular substrates involved in different signalling cascades. They are also involved in regulating pathogen life cycle and infectivity. In this review, we attempt to address the role of parasite kinome in host infection, pathogen survival within the host tissue and thereby disease manifestation. The understanding of the parasite kinome can be a potential target for robust diagnosis and effective therapeutics.

A suicide inhibitor of nematode trehalose-6-phosphate phosphatases.

Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 µM and 0.56 µM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber's pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 µM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.

The anti-phytoalexin gene Bx-cathepsin W supports the survival of Bursaphelenchus xylophilus under Pinus massoniana phytoalexin stress.

BACKGROUND: Pine trees challenged by Bursaphelenchus xylophilus invasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective against B. xylophilus. The anti-phytoalexin genes of B. xylophilus disable almost all Asian pine phytoalexins, which has allowed B. xylophilus to devastate pine forests in eastern Asia over the last four decades. However, to date, the factors that stimulate anti-phytoalexin gene expression and the mechanisms by which these genes act are not well understood. RESULTS: Here, we described anti-phytoalexin genes in B. xylophilus using transcriptomic and bioinformatics analyses. The genes that were induced by both Pinus massoniana and carvone and had similarly elevated expression trends were considered anti-phytoalexin genes. Altogether, 187 anti-phytoalexin genes were identified, including 4 cathepsin genes. KEGG pathway enrichment indicated that those cathepsins were related to the Lysosome pathway. Since cathepsins help to maintain metabolic homeostasis by participating in the degradation of heterophagic and autophagic material, the lysosomal cathepsin gene Bx-cathepsin W was cloned and characterized. The results of the RNAi assessment indicated that the knockdown of Bx-cathepsin W reduced the survival rates of B. xylophilus under carvone or P. massoniana stress. The correlation between Bx-cathepsin W and the susceptibility of pines showed that Bx-cathepsin W might help improve the anti-phytotoxin ability of B. xylophilus. CONCLUSIONS: The results indicated that the anti-phytoalexin gene Bx-cathepsin W supported the survival of B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.

Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices.

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed ß-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp-Glu-Ser), Nematoda (Asp-Asp-His) and Echinodermata (Asp-Glu-Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

Episodic evolution of a eukaryotic NADK repertoire of ancient provenance.

NAD kinase (NADK) is the sole enzyme that phosphorylates nicotinamide adenine dinucleotide (NAD+/NADH) into NADP+/NADPH, which provides the chemical reducing power in anabolic (biosynthetic) pathways. While prokaryotes typically encode a single NADK, eukaryotes encode multiple NADKs. How these different NADK genes are all related to each other and those of prokaryotes is not known. Here we conduct phylogenetic analysis of NADK genes and identify major clade-defining patterns of NADK evolution. First, almost all eukaryotic NADK genes belong to one of two ancient eukaryotic sister clades corresponding to cytosolic ("cyto") and mitochondrial ("mito") clades. Secondly, we find that the cyto-clade NADK gene is duplicated in connection with loss of the mito-clade NADK gene in several eukaryotic clades or with acquisition of plastids in Archaeplastida. Thirdly, we find that horizontal gene transfers from proteobacteria have replaced mitochondrial NADK genes in only a few rare cases. Last, we find that the eukaryotic cyto and mito paralogs are unrelated to independent duplications that occurred in sporulating bacteria, once in mycelial Actinobacteria and once in aerobic endospore-forming Firmicutes. Altogether these findings show that the eukaryotic NADK gene repertoire is ancient and evolves episodically with major evolutionary transitions.

Nematode Chitin and Application.

Plant-parasitic nematode infection is a global problem for agriculture and forestry. There is clearly a need for novel nematicides, because of the pitifully small repertoire of nematicides available and the effectiveness of losing or environmental prohibition of these nematicides. Chitin is the essential component of nematode eggshell and pharynx. The disturbance of chitin synthesis or hydrolysis led to nematode embryonic lethal, laying defective eggs or moulting failure. Thus, the key components in the chitin metabolic process are promising targets for anti-nematode agent's development. In this chapter, we focus on chitin and chitin synthase of nematodes, chitinases and their roles in nematode survival and application of chitin in nematode control.

Cysteine proteases secreted by the pinewood nematode, Bursaphelenchus xylophilus: In silico analysis.

The pinewood nematode, Bursaphelenchus xylophilus, is an important plant-parasitic nematode responsible for the development of the pine wilt disease and recognised as a major forest pest. Previous studies on the comparison of B. xylophilus and B. mucronatus secretomes obtained under maritime pine, Pinus pinaster, wood extract stimulus revealed that several cysteine proteases were increased in B. xylophilus secretome. In nematodes, proteases are known to play critical roles in parasitic processes like tissue penetration, digestion of host tissues for nutrition and evasion of host immune response. To gain further insight into the possible role of cysteine proteases on B. xylophilus pathogenicity, the molecular characterisation of four secreted cysteine peptidases was performed. BxCP3 and BxCP11 were identified as cathepsin L-like proteins and BxCP7 and BxCP8 as cathepsin B proteins. Only BxCP8 revealed high homology with another B. xylophilus cathepsin B referred on GenBank, all the others differ from the closer proteins deposited in this database. In silico three-dimensional structures of the four BxCP suggest that these proteins are pro-enzymes that become active when the pro-peptide is cleaved. BxCP7 and BxCP8 predicted structures revealed the presence of an occluding loop that occludes the active site cleft, typical of cathepsin B proteases.

A Novel Antithrombotic Protease from Marine Worm Sipunculus Nudus.

Sipunculus nudus, an old marine species, has great potential for use as functional seafood due to its various bioactivities. Its potential antithrombotic activity pushed us to isolate the bio-active components bio-guided by tracking fibrinolytic activity. As a result, a novel protease named as SK (the kinase obtained from S. nudus) was obtained, which possessed a molecular weight of 28,003.67 Da and 15 N-terminal amino acid sequences of PFPVPDPFVWDTSFQ. SK exerted inhibitory effects on thrombus formation through improving the coagulation system with dose-effect relationship within a certain range. Furthermore, in most cases SK got obviously better effect than that of urokinase. With the help of untargeted mass spectrometry-based metabolomics profiling, arachidonic acid, sphingolipid, and nicotinate and nicotinamide mechanism pathways were found to be important pathways. They revealed that the effect mechanism of SK on common carotid arterial thrombosis induced by FeCl3 was achieved by inhibiting vessel contraction, platelet aggregation, adhesion, and release, correcting endothelial cell dysfunction and retarding process of thrombus formation. This study demonstrated SK was a promising thrombolytic agent on the basis of its comprehensive activities on thrombosis, and it should get further exploitation and utilization.

Small Molecule Inhibitors of Metabolic Enzymes Repurposed as a New Class of Anthelmintics.

The enormous prevalence of infections caused by parasitic nematodes worldwide, coupled to the rapid emergence of their resistance to commonly used anthelmintic drugs, presents an urgent need for the discovery of new drugs. Herein, we have identified several classes of small molecules with broad spectrum activity against these pathogens. Previously, we reported the identification of carnitine palmitoyltransferases (CPTs) as a representative class of enzymes as potential targets for metabolic chokepoint intervention that was elucidated from a combination of chemogenomic screening and experimental testing in nematodes. Expanding on these previous findings, we have discovered that several chemical classes of known small molecule inhibitors of mammalian CPTs have potent activity as anthelmintics. Cross-clade efficacy against a broad spectrum of adult parasitic nematodes was demonstrated for multiple compounds from different series. Several analogs of these initial hit compounds were designed and synthesized. The compounds we report represent a good starting point for further lead identification and optimization for development of new anthelmintic drugs with broad spectrum activity and a novel mechanism of action.

Cloning and expression analysis of the DEAD-box/RNA helicase Oslaf-1 in Ovomermis sinensis.

Ovomermis sinensis is a potentially-valuable nematode for controlling insect pests. The parasitic stage of the nematode absorbs nutrients in its host's hemolymph to maintain its growth development and then kills the host when it emerges. At present, little known about its reproductive development, particularly the responsible molecular mechanism. More detailed research on the genes of reproductive development will not only help us understand the mechanisms underlying sex differentiation in the nematode, but would also be valuable for successfully cultivating them in vitro and using them for biocontrol. In this study, we used the homology cloning method to clone the full-length cDNA of a DEAD-box family gene (Oslaf-1) from O. sinensis. Then, using qRT-PCR technology to detect the expression pattern of the Oslaf-1 gene at different development stages and tissues, the gene was found to be highly expressed in the post-parasitic stage (P < 0.01) and ovarian (P < 0.05) of O. sinensis. Western blot analysis showed the same result that the gene is associated with gonadal development and function, but is not gonad-specific. In situ hybridization further demonstrated that the gene is widely expressed in early embryos and is mainly distributed in the gonadal area. However, the signal was mainly concentrated in the reproductive primordia in pre-parasitic juveniles. RNA interference (RNAi) studies revealed that the sex ratio of O. sinensis soaked in dsRNA of Oslaf-1 was not statistically different than the gfp dsRNA treated groups. Our results suggest that Oslaf-1 may play a vital role in the reproductive systems of the nematode. In addition, we speculate that the Oslaf-1 gene plays an important role during embryonic development and that it occurs and develops in the gonads of pre-parasitic juveniles of O. sinensis.

A clustering package for nucleotide sequences using Laplacian Eigenmaps and Gaussian Mixture Model.

In this article, a new Python package for nucleotide sequences clustering is proposed. This package, freely available on-line, implements a Laplacian eigenmap embedding and a Gaussian Mixture Model for DNA clustering. It takes nucleotide sequences as input, and produces the optimal number of clusters along with a relevant visualization. Despite the fact that we did not optimise the computational speed, our method still performs reasonably well in practice. Our focus was mainly on data analytics and accuracy and as a result, our approach outperforms the state of the art, even in the case of divergent sequences. Furthermore, an a priori knowledge on the number of clusters is not required here. For the sake of illustration, this method is applied on a set of 100 DNA sequences taken from the mitochondrially encoded NADH dehydrogenase 3 (ND3) gene, extracted from a collection of Platyhelminthes and Nematoda species. The resulting clusters are tightly consistent with the phylogenetic tree computed using a maximum likelihood approach on gene alignment. They are coherent too with the NCBI taxonomy. Further test results based on synthesized data are then provided, showing that the proposed approach is better able to recover the clusters than the most widely used software, namely Cd-hit-est and BLASTClust.

Identification and characterization of a novel ß-glucosidase via metagenomic analysis of Bursaphelenchus xylophilus and its microbial flora.

ß-glucosidases catalyze the final step of cellulose hydrolysis and are essential in cellulose degradation. A ß-glucosidase gene, cen502, was identified and isolated from a metagenomic library from Bursaphelenchus xylophilus via functional screening. Analyses indicated that cen502 encodes a 465 amino acid polypeptide that contains a catalytic domain belonging to the glycoside hydrolase family 1 (GH1). Cen502 was heterologously expressed, purified, and biochemically characterized. Recombinant Cen502 displayed optimum enzymatic activity at pH 8.0 and 38 °C. The enzyme had highest specific activity to p-nitrophenyl-ß-D-glucopyranoside (pNPG; 180.3 U/mg) and had K m and V max values of 2.334 mol/ml and 9.017 µmol/min/mg, respectively. The addition of Fe2+ and Mn2+ significantly increased Cen502 ß-glucosidase activity by 60% and 50%, respectively, while 10% and 25% loss of ß-glucosidase activity was induced by addition of Pb2+ and K+, respectively. Cen502 exhibited activity against a broad array of substrates, including cellobiose, lactose, salicin, lichenan, laminarin, and sophorose. However, Cen502 displayed a preference for the hydrolysis of ß-1,4 glycosidic bonds rather than ß-1,3, ß-1,6, or ß-1,2 bonds. Our results indicate that Cen502 is a novel ß-glucosidase derived from bacteria associated with B. xylophilus and may represent a promising target to enhance the efficiency of cellulose bio-degradation in industrial applications.

Molecular characterization and functional analysis of a glutathione peroxidase gene from Aphelenchoides besseyi (Nematoda: Aphelenchoididae).

Aphelenchoides besseyi, the nematode agent of rice tip white disease, causes huge economic losses in almost all the rice-growing regions of the world. Glutathione peroxidase (GPx), an esophageal glands secretion protein, plays important roles in the parasitism, immune evasion, reproduction and pathogenesis of many plant-parasitic nematodes (PPNs). Therefore, GPx is a promising target for control A. besseyi. Here, the full-length sequence of the GPx gene from A. besseyi (AbGPx1) was cloned using the rapid amplification of cDNA ends method. The full-length 944 bp AbGPx1 sequence, which contains a 678 bp open reading frame, encodes a 225 amino acid protein. The deduced amino acid sequence of the AbGPxl shares highly homologous with other nematode GPxs, and showed the closest evolutionary relationship with DrGPx. In situ hybridization showed that AbGPx1 was constitutively expressed in the esophageal glands of A. besseyi, suggesting its potential roles in parasitism and reproduction. RNA interference (RNAi) was used to assess the functions of the AbGPx1 gene, and quantitative real-time PCR was used to monitor the RNAi effects. After treatment with dsRNA for 12 h, AbGPx1 expression levels and reproduction in the nematodes decreased compared with the same parameters in the control group; thus, the AbGPx1 gene is likely to be associated with the development, reproduction, and infection ability of A. besseyi. These findings may open new avenues towards nematode control.

Enzyme characteristics of pathogen-specific trehalose-6-phosphate phosphatases.

Owing to the key role of trehalose in pathogenic organisms, there has recently been growing interest in trehalose metabolism for therapeutic purposes. Trehalose-6-phosphate phosphatase (TPP) is a pivotal enzyme in the most prominent biosynthesis pathway (OtsAB). Here, we compare the enzyme characteristics of recombinant TPPs from five important nematode and bacterial pathogens, including three novel members of this protein family. Analysis of the kinetics of trehalose-6-phosphate hydrolysis reveals that all five enzymes display a burst-like kinetic behaviour which is characterised by a decrease of the enzymatic rate after the pre-steady state. The observed super-stoichiometric burst amplitudes can be explained by multiple global conformational changes in members of this enzyme family during substrate processing. In the search for specific TPP inhibitors, the trapping of the complex conformational transitions in TPPs during the catalytic cycle may present a worthwhile strategy to explore.

Acetylcholinesterases from entomopathogenic nematode Heterorhabditid bacteriophora: Susceptibility to insecticides and immunological characteristics.

Acetylcholinesterases (AChEs) from the infective juveniles (IJs) of entomopathogenic nematode (EPN) have been investigated with respect to their susceptibility to insecticides and immunological characteristics, aiming at nominating the most compatible insecticide(s) to be used in conjunction with the most insecticide-tolerant EPN strain before incorporation in integrated pest management (IPM) programs. The inhibition kinetics of two purified AChE isoenzymes, AChEAII and AChEBI isolated from Heterorhabditid bacteriophora EM2 strain, by different insecticides revealed that the insensitivity to inhibition by such insecticides could be arranged in a descending order as; methomyl>carbofuran>acetamiprid>oxamyl>malathion. Except for malathion, the insecticides competitively inhibited AChEs with Ki values ranging from 0.1 to 15mM and IC50 values from 1.25 to 23mM. The two AChE isoforms are several folds less sensitive to inhibition by methomyl and carbofuran compared to those previously reported for other insect species. AChEBI was used as an immunogen to raise anti-AChEBI antisera in rabbits. The prepared antisera cross-reacted with AChEs of five different heterorhabditid nematode strains implying the presence of common epitopes shared along all the examined strains. Such studies could aid in the rational selection of the compatible insecticide(s) and the prepared polyclonal anti-AChE antisera would be a valuable immunodiagnostic tool for evaluating the most insecticide-tolerant EPN strain(s) in IPM programs.