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1.
PLoS Negl Trop Dis ; 14(8): e0008517, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810153

RESUMO

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.


Assuntos
Anti-Helmínticos/farmacologia , Oxamniquine/análogos & derivados , Oxamniquine/farmacologia , Schistosoma/efeitos dos fármacos , Esquistossomose/parasitologia , Animais , Anti-Helmínticos/química , Simulação por Computador , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Oxamniquine/química , Ligação Proteica
2.
PLoS One ; 15(8): e0237150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760111

RESUMO

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.


Assuntos
Antinematódeos/farmacologia , Dirofilaria immitis/efeitos dos fármacos , Ivermectina/farmacologia , Macrolídeos/farmacologia , Fenótipo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Dirofilaria immitis/metabolismo , Dirofilaria immitis/patogenicidade , Dirofilariose/parasitologia , Cães , Resistência a Medicamentos , Proteínas de Helminto/metabolismo
3.
PLoS Negl Trop Dis ; 14(7): e0008470, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644998

RESUMO

BACKGROUND: Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. METHODOLOGY/PRINCIPAL FINDINGS: We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34-117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34-117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34-117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34-117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to inflammatory responses. CONCLUSIONS/SIGNIFICANCE: These results offer new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, and places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities.


Assuntos
Antígenos de Helmintos/metabolismo , Proteínas de Helminto/metabolismo , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Células da Medula Óssea , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Humanos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óvulo , Filogenia , Transporte Proteico
4.
PLoS Negl Trop Dis ; 14(7): e0008447, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730343

RESUMO

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.


Assuntos
Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Helminto/metabolismo , Diester Fosfórico Hidrolases/genética , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Animais , Linhagem Celular , Deleção de Genes , Perfilação da Expressão Gênica , Genoma Helmíntico , Proteínas de Helminto/genética , Masculino , Camundongos , Filogenia , Trypanosoma brucei brucei , Leveduras
5.
Parasitol Res ; 119(7): 2217-2226, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32500370

RESUMO

Schistosoma is the causative agent of schistosomiasis, a common infectious disease distributed worldwide. Our previous phosphoproteomic analysis suggested that glycogen synthase kinase 3 (GSK3), a conserved protein kinase in eukaryotes, is likely involved in protein phosphorylation of Schistosoma japonicum. Here, we aimed to identify the interacting partners of S. japonicum GSK3ß (SjGSK3ß) and to evaluate its role in parasite survival. Toward these ends, we determined the transcription levels of SjGSK3ß at different developmental stages and identified its interacting partners of SjGSK3ß by screening a yeast two-hybrid S. japonicum cDNA library. We further used RNA interference (RNAi) to inhibit the expression of SjGSK3ß in adult worms in vitro and examined the resultant changes in transcription of its putative interacting proteins and in worm viability compared with those of control worms. Reverse transcription-quantitative polymerase chain analysis indicated that SjGSK3ß is expressed throughout the life cycle of S. japonicum, with higher expression levels detected in the eggs and relatively higher expression level found in male worms than in female worms. By screening the yeast two-hybrid library, eight proteins were identified as potentially interacting with SjGSK3ß including cell division cycle 37 homolog (Cdc37), 14-3-3 protein, tegument antigen (I(H)A), V-ATPase proteolipid subunit, myosin alkali light chain 1, and three proteins without recognized functional domains. In addition, SjGSK3ß RNAi reduced the SjGSK3ß gene transcript level, leading to a significant decrease in kinase activity, cell viability, and worm survival. Collectively, these findings suggested that SjGSK3ß may interact with its partner proteins to influence worm survival by regulating kinase activity.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Helminto/metabolismo , Schistosoma japonicum/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Helminto/genética , Masculino , Ligação Proteica , Interferência de RNA , Schistosoma japonicum/enzimologia , Schistosoma japonicum/genética , Análise de Sobrevida , Técnicas do Sistema de Duplo-Híbrido
6.
PLoS Biol ; 18(6): e3000723, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511224

RESUMO

Lymphatic filariasis (LF) afflicts over 60 million people worldwide and leads to severe pathological outcomes in chronic cases. The nematode parasites (Nematoda: Filarioidea) that cause LF require both arthropod (mosquito) intermediate hosts and mammalian definitive hosts for their propagation. The invasion and migration of filarial worms through host tissues are complex and critical to survival, yet little is known about the receptors and signaling pathways that mediate directed migration in these medically important species. In order to better understand the role of chemosensory signaling in filarial worm taxis, we employ comparative genomics, transcriptomics, reverse genetics, and chemical approaches to identify putative chemosensory receptor proteins and perturb chemotaxis phenotypes in filarial worms. We find that chemoreceptor family size is correlated with the presence of environmental (extrahost) stages in nematode life cycles, and that filarial worms contain compact and highly diverged chemoreceptor complements and lineage-specific ion channels that are predicted to operate downstream of chemoreceptor activation. In Brugia malayi, an etiological agent of LF, chemoreceptor expression patterns correspond to distinct parasite migration events across the life cycle. To interrogate the role of chemosensation in the migration of larval worms, arthropod and mammalian infectious stage Brugia parasites were incubated in nicotinamide, an agonist of the nematode transient receptor potential (TRP) channel OSM-9. Exposure of microfilariae to nicotinamide alters intramosquito migration, and exposure of L3s reduces chemotaxis toward host-associated cues in vitro. Nicotinamide also potently modulates thermosensory responses in L3s, suggesting a polymodal sensory role for Brugia osm-9. Reverse genetic studies implicate both Brugia osm-9 and the cyclic nucleotide-gated (CNG) channel subunit tax-4 in larval chemotaxis toward host serum, and these ion channel subunits partially rescue sensory defects in Caenorhabditis elegans osm-9 and tax-4 knock-out strains. Together, these data reveal genetic and functional diversification of chemosensory signaling proteins in filarial worms and encourage a more thorough investigation of clade- and parasite-specific facets of nematode sensory receptor biology.


Assuntos
Brugia Malayi/genética , Células Quimiorreceptoras/metabolismo , Culicidae/parasitologia , Filariose Linfática/parasitologia , Variação Genética , Animais , Caenorhabditis elegans/fisiologia , Quimiotaxia , Genoma , Proteínas de Helminto/metabolismo , Larva , Estágios do Ciclo de Vida , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo , Temperatura
7.
Nat Commun ; 11(1): 2674, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471987

RESUMO

Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs) at inhibitory neuromuscular junctions. Here we show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de GABA-A/metabolismo , Transmissão Sináptica/fisiologia , Animais , Orientação de Axônios/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Sinapses/fisiologia
8.
PLoS One ; 15(5): e0233048, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453791

RESUMO

Panagrolaimus sp. DAW1, a nematode cultured from the Antarctic, has the extraordinary physiological ability to survive total intracellular freezing throughout all of its compartments. While a few other organisms, all nematodes, have subsequently also been found to survive freezing in this manner, P. sp. DAW1 has so far shown the highest survival rates. In addition, P. sp. DAW1 is also, depending on the rate or extent of freezing, able to undergo cryoprotective dehydration. In this study, the proteome of P. sp DAW1 is explored, highlighting a number of differentially expressed proteins and pathways that occur when the nematodes undergo intracellular freezing. Among the strongest signals after being frozen is an upregulation of proteases and the downregulation of cytoskeletal and antioxidant activity, the latter possibly accumulated before freezing much in the way the sugar trehalose has been shown to be stored during acclimation.


Assuntos
Aclimatação/fisiologia , Redes Reguladoras de Genes , Proteômica/métodos , Rabditídios/fisiologia , Animais , Antioxidantes/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica , Proteínas de Helminto/metabolismo , Peptídeo Hidrolases/metabolismo , Mapas de Interação de Proteínas
9.
Parasitol Res ; 119(7): 2159-2176, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32424554

RESUMO

The proteasome is the key player in the cellular protein degradation machinery and is pivotal for protein homeostasis and Schistosoma mansoni (S. mansoni) survival. Our group study provides insights into proteasome inhibitors and reveals that selective schistosomiasis agents represent an interesting branch of proteasome research linked to the development of new drugs for this neglected disease. Here, we explored the phenotypic response of S. mansoni to b-AP15, a bis-benzylidine piperidone that inhibits 26S proteasome deubiquitinases (DUBs), ubiquitin-specific protease 14 (USP14), and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5). b-AP15 induces a modest decrease in egg production in vitro and reduces viability, leading to the death of parasite couples. This inhibitor also induces a twofold increase in the accumulation of polyubiquitinated proteins in S. mansoni adult worms and causes tegument changes such as disintegration, wrinkling, and bubble formation, both throughout the length of the parasite and in the oral sucker. b-AP15 alters the cell organelles of adult S. mansoni worms, and we specifically observed mitochondrial alterations, which are suggestive of proteotoxic stress leading to autophagy. Taken together, these results indicate that the deubiquitinase function of the proteasome is essential for the parasite and support the hypothesis that the proteasome constitutes an interesting drug target for the treatment of schistosomiasis.


Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Oviposição/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Feminino , Proteínas de Helminto/metabolismo , Piperidonas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Schistosoma mansoni/metabolismo , Schistosoma mansoni/fisiologia , Ubiquitinação/efeitos dos fármacos
10.
PLoS Negl Trop Dis ; 14(5): e0007743, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32374726

RESUMO

Schistosomes are parasitic blood flukes that survive for many years within the mammalian host vasculature. How the parasites establish a chronic infection in the hostile bloodstream environment, whilst evading the host immune response is poorly understood. The parasite develops morphologically and grows as it migrates to its preferred vascular niche, avoiding or repairing damage from the host immune system. In this study, we investigated temporal changes in gene expression during the intra-mammalian development of Schistosoma mansoni. RNA-seq data were analysed from parasites developing in the lung through to egg-laying mature adult worms, providing a comprehensive picture of in vivo intra-mammalian development. Remarkably, genes involved in signalling pathways, developmental control, and adaptation to oxidative stress were up-regulated in the lung stage. The data also suggested a potential role in immune evasion for a previously uncharacterised gene. This study not only provides a large and comprehensive data resource for the research community, but also reveals new directions for further characterising host-parasite interactions that could ultimately lead to new control strategies for this neglected tropical disease pathogen.


Assuntos
Proteínas de Helminto/genética , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/genética , Esquistossomose mansoni/parasitologia , Animais , Feminino , Proteínas de Helminto/metabolismo , Humanos , Masculino , Camundongos , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/patologia , Transcriptoma
11.
PLoS Pathog ; 16(4): e1008465, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271834

RESUMO

Roundworm parasite infections are a major cause of human and livestock disease worldwide and a threat to global food security. Disease control currently relies on anthelmintic drugs to which roundworms are becoming increasingly resistant. An alternative approach is control by vaccination and 'hidden antigens', components of the worm gut not encountered by the infected host, have been exploited to produce Barbervax, the first commercial vaccine for a gut dwelling nematode of any host. Here we present the structure of H-gal-GP, a hidden antigen from Haemonchus contortus, the Barber's Pole worm, and a major component of Barbervax. We demonstrate its novel architecture, subunit composition and topology, flexibility and heterogeneity using cryo-electron microscopy, mass spectrometry, and modelling. Importantly, we demonstrate that complexes with the same architecture are present in other Strongylid roundworm parasites including human hookworm. This suggests a common ancestry and the potential for development of a unified hidden antigen vaccine.


Assuntos
Endopeptidases/metabolismo , Endopeptidases/ultraestrutura , Haemonchus/imunologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Animais , Anti-Helmínticos/farmacologia , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/imunologia , Microscopia Crioeletrônica , Endopeptidases/imunologia , Haemonchus/patogenicidade , Proteínas de Helminto/imunologia , Glicoproteínas de Membrana/imunologia , Parasitos , Vacinação , Vacinas/imunologia
12.
Parasitol Res ; 119(6): 1777-1784, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32300877

RESUMO

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.


Assuntos
Ancylostoma/química , Proliferação de Células/efeitos dos fármacos , Proteínas de Helminto/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Ancylostoma/genética , Animais , Citocinas/metabolismo , Cães , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Inibidores da Agregação de Plaquetas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
13.
PLoS Genet ; 16(4): e1008687, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282814

RESUMO

Environment shapes development through a phenomenon called developmental plasticity. Deciphering its genetic basis has potential to shed light on the origin of novel traits and adaptation to environmental change. However, molecular studies are scarce, and little is known about molecular mechanisms associated with plasticity. We investigated the gene regulatory network controlling predatory vs. non-predatory dimorphism in the nematode Pristionchus pacificus and found that it consists of genes of extremely different age classes. We isolated mutants in the conserved nuclear hormone receptor nhr-1 with previously unseen phenotypic effects. They disrupt mouth-form determination and result in animals combining features of both wild-type morphs. In contrast, mutants in another conserved nuclear hormone receptor nhr-40 display altered morph ratios, but no intermediate morphology. Despite divergent modes of control, NHR-1 and NHR-40 share transcriptional targets, which encode extracellular proteins that have no orthologs in Caenorhabditis elegans and result from lineage-specific expansions. An array of transcriptional reporters revealed co-expression of all tested targets in the same pharyngeal gland cell. Major morphological changes in this gland cell accompanied the evolution of teeth and predation, linking rapid gene turnover with morphological innovations. Thus, the origin of feeding plasticity involved novelty at the level of genes, cells and behavior.


Assuntos
Evolução Molecular , Proteínas de Helminto/genética , Comportamento Predatório , Receptores Citoplasmáticos e Nucleares/genética , Rabditídios/genética , Animais , Sequência Conservada , Redes Reguladoras de Genes , Proteínas de Helminto/metabolismo , Boca/anatomia & histologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Rabditídios/anatomia & histologia , Rabditídios/fisiologia , Análise de Célula Única
14.
PLoS Pathog ; 16(4): e1008396, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32243475

RESUMO

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.


Assuntos
Antinematódeos/farmacologia , Ascaris suum/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Faringe/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Ascaris suum/efeitos dos fármacos , Ascaris suum/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Helminto/genética , Faringe/efeitos dos fármacos , Receptores Nicotínicos/genética
15.
Res Vet Sci ; 130: 110-117, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32171999

RESUMO

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis.


Assuntos
Catepsina B/genética , Enterócitos/parasitologia , Células Epiteliais/parasitologia , Proteínas de Helminto/genética , Mucosa Intestinal/parasitologia , Trichinella spiralis/fisiologia , Animais , Catepsina B/metabolismo , Feminino , Proteínas de Helminto/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência de DNA/veterinária , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/parasitologia
16.
PLoS Negl Trop Dis ; 14(3): e0008115, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203512

RESUMO

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a 'footprint' of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.


Assuntos
Proteínas de Helminto/metabolismo , Fosfoproteínas/metabolismo , Proteoma/análise , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Feminino , Proteínas de Helminto/genética , Masculino , Peptídeos/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteínas Quinases , Processamento de Proteína Pós-Traducional , Schistosoma mansoni/genética , Transdução de Sinais
17.
Biochem Biophys Res Commun ; 524(2): 411-417, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32005520

RESUMO

As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type Ⅰ PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian.


Assuntos
Proteínas de Helminto/metabolismo , Planárias/fisiologia , Proteína-Arginina N-Metiltransferases/metabolismo , Regeneração , Sequência de Aminoácidos , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Proteínas de Helminto/química , Proteínas de Helminto/genética , Insetos , Planárias/genética , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , RNA Mensageiro/genética , Alinhamento de Sequência
18.
PLoS Genet ; 16(2): e1008613, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32078629

RESUMO

The extracellular matrix (ECM) is important for maintaining the boundaries between tissues. This role is particularly critical in the stem cell niche, as pre-neoplastic or cancerous stem cells must pass these boundaries in order to invade into the surrounding tissue. Here, we examine the role of the ECM as a regulator of the stem cell compartment in the planarian Schmidtea mediterranea, a highly regenerative, long-lived organism with a large population of adult stem cells. We identify two EGF repeat-containing genes, megf6 and hemicentin, with identical knockdown phenotypes. We find that megf6 and hemicentin are needed to maintain the structure of the basal lamina, and in the absence of either gene, pluripotent stem cells migrate ectopically outside of their compartment and hyper-proliferate, causing lesions in the body wall muscle. These muscle lesions and ectopic stem cells are also associated with ectopic gut branches, which protrude from the normal gut towards the dorsal side of the animal. Interestingly, both megf6 and hemicentin knockdown worms are capable of regenerating tissue free of both muscle lesions and ectopic cells, indicating that these genes are dispensable for regeneration. These results provide insight into the role of planarian ECM in restricting the stem cell compartment, and suggest that signals within the compartment may act to suppress stem cell hyperproliferation.


Assuntos
Células-Tronco Adultas/fisiologia , Genes de Helmintos/fisiologia , Platelmintos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Nicho de Células-Tronco/genética , Animais , Animais Geneticamente Modificados , Movimento Celular/genética , Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Platelmintos/citologia , Regeneração/genética
19.
Parasitol Int ; 75: 102050, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31901435

RESUMO

The carcinogenic liver fluke Opisthorchis viverrini (O. viverrini) is endemic in Thailand and neighboring countries including Laos PDR, Vietnam and Cambodia. Infections with O. viverrini lead to hepatobiliary abnormalities including bile duct cancer-cholangiocarcinoma (CCA). Despite decades of extensive studies, the underlying mechanisms of how this parasite survives in the bile duct and causes disease are still unclear. Therefore, this study aims to identify and characterize the most abundant protein secreted by the parasite. Proteomics and bioinformatics analysis revealed that the most abundant secretory protein is a metallopeptidase, named Ov-M60-like-1. This protein contains an N-terminal carbohydrate-binding domain and a C-terminal M60-like domain with a zinc metallopeptidase HEXXH motif. Further analysis by mass spectrometry revealed that Ov-M60-like-1 is N-glycosylated. Recombinant Ov-M60-like-1 (rOv-M60-like-1) expressed in Escherichia coli (E. coli) was able to digest bovine submaxillary mucin (BSM). The mucinase activity was inhibited by the ion chelating agent EDTA, confirming its metallopeptidase identity. The enzyme was active at temperatures ranging 25-37 °C in a broad pH range (pH 2-10). The identification of Ov-M60-like-1 mucinase as the major secretory protein of O. viverrini worms warrants further research into the role of this glycoprotein in the pathology induced by this carcinogenic worm.


Assuntos
Proteínas de Helminto/genética , Metaloproteases/genética , Opisthorchis/genética , Sequência de Aminoácidos , Animais , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Metaloproteases/química , Metaloproteases/metabolismo , Opistorquíase/metabolismo , Opisthorchis/enzimologia , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
20.
J Agric Food Chem ; 68(2): 523-529, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31908169

RESUMO

The present work sought to contribute to the development of new nematicides. Benzaldehydes were initially converted to nitrile oxides that underwent 1,3-dipolar cycloaddition reactions with methyl acrylate to generate 4,5-dihydroisoxazoles. In in vitro tests, methyl 3-phenyl-4,5-dihydroisoxazole-5-carboxylate (1) and methyl 3-(4-chlorophenyl)-4,5-dihydroisoxazole-5-carboxylate (4) increased the mortality of Meloidogyne exigua and Meloidogyne incognita second-stage juveniles (J2). Compounds 1 and 4 presented necessary concentrations of 398 and 501 µg mL-1, respectively, to kill 50% of M. incognita J2 (LC50 values), while the value for carbofuran (positive control) was 168 µg mL-1. In in vivo tests, compounds 1 and 4 reduced the number of M. incognita galls in tomato roots by 70 and 40%, respectively, and the number of eggs by 89 and 44%. Using an in silico approach, we showed that compounds 1 and 4 were toxic to the nematodes by binding to the allosteric binding sites of the agonist-binding domains of the nematode nicotinic acetylcholine receptors. These results opened up possibilities for further investigations aimed at developing novel commercial nematicides.


Assuntos
Antinematódeos/toxicidade , Isoxazóis/toxicidade , Doenças das Plantas/parasitologia , Tylenchoidea/efeitos dos fármacos , Animais , Antinematódeos/química , Simulação por Computador , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Isoxazóis/química , Lycopersicon esculentum/parasitologia , Raízes de Plantas/parasitologia , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Tylenchoidea/crescimento & desenvolvimento , Tylenchoidea/metabolismo
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