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1.
Gene ; 775: 145440, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33482282

RESUMO

Tubgcp3/GCP3 (The centrosomal protein γ-tubulin complex protein 3) is a component of the γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), which play critical roles in mitotic spindle formation during mitosis. However, its function in stem cell development has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is a unique model to study stem cell lineage development in vivo. Here, we identified a homolog of Tubgcp3 in planarian Dugesia japonica, and found that Tubgcp3 is required for the maintenance of epidermal lineage. RNAi targeting Tubgcp3 resulted in tissue homeostasis and regeneration defect. Knockdown of Tubgcp3 reduced cell divisions and led to a loss of the mature epidermal cells. Our findings indicate that Tubgcp3 is a mitotic regulator and plays a crucial role in planarian epidermal differentiation.


Assuntos
Epiderme/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Animais , Diferenciação Celular , Proliferação de Células , Clonagem Molecular , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Planárias , Regeneração
2.
Korean J Parasitol ; 58(5): 513-525, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33202503

RESUMO

Clonorchis sinensis is a food-borne trematode that infects more than 15 million people. The liver fluke causes clonorchiasis and chronical cholangitis, and promotes cholangiocarcinoma. The underlying molecular pathogenesis occurring in the bile duct by the infection is little known. In this study, transcriptome profile in the bile ducts infected with C. sinensis were analyzed using microarray methods. Differentially expressed genes (DEGs) were 1,563 and 1,457 at 2 and 4 weeks after infection. Majority of the DEGs were temporally dysregulated at 2 weeks, but 519 DEGs showed monotonically changing expression patterns that formed seven distinct expression profiles. Protein-protein interaction (PPI) analysis of the DEG products revealed 5 sub-networks and 10 key hub proteins while weighted co-expression network analysis (WGCNA)-derived gene-gene interaction exhibited 16 co-expression modules and 13 key hub genes. The DEGs were significantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes pathways, which were related to original systems, cellular process, environmental information processing, and human diseases. This study uncovered a global picture of gene expression profiles in the bile ducts infected with C. sinensis, and provided a set of potent predictive biomarkers for early diagnosis of clonorchiasis.


Assuntos
Ductos Biliares/patologia , Ductos Biliares/parasitologia , Clonorquíase/diagnóstico , Clonorquíase/parasitologia , Clonorchis sinensis/genética , Expressão Gênica/genética , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Transdução de Sinais/genética , Transcriptoma , Animais , Diagnóstico Precoce , Epistasia Genética , Ratos Sprague-Dawley
3.
Parasitol Res ; 119(11): 3705-3718, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32901341

RESUMO

Excretory-secretory products (ESPs) of parasitic helminths are well known to exert immunostimulation and immunomodulation in hosts. Immune regulation plays a key role in anti-tumour therapy. The present study explored the anti-tumour effect of ESPs released by Angiostrongylus cantonensis. In Hepa1-6 mouse tumour models, ESPs significantly reduced tumour growth. Tumour-bearing mice treated with ESPs had significantly higher CD3+, CD4+, and CD8+ T cell counts than those treated with Freund's adjuvant. In vitro, human hepatocarcinoma HepG2 cells, human lung cancer A549 cells, and normal human liver HL-7702 cells were co-incubated with ESPs for 24 h and 48 h. ESPs significantly accelerated HepG2 apoptosis but had no inhibitory effect on the proliferation of A549 and HL-7702 cells. Apoptotic HepG2 cells displayed condensed nuclei, apoptotic bodies, and swollen endoplasmic reticulum (ER). Expression of the endoplasmic reticulum stress (ERS)-related factors activating transcription factor 6 (ATF6) and C/EBP-homologous protein (CHOP) in HepG2 cells increased with increasing ESP concentration and treatment time. Calreticulin (CRT) is a key effector protein of ESPs, and recombinant calreticulin (rCRT) was produced in BL21 Escherichia coli (E. coli). In contrast to ESPs, rCRT markedly reduced the proliferation of HepG2 cells. The expression levels of ATF6 and CHOP in HepG2 cells treated with 30 µg/mL rCRT significantly increased at 48 h. Notably, these findings synergistically suggest that ESPs and rCRT are promising candidates for anti-tumour immunotherapy.


Assuntos
Angiostrongylus cantonensis/metabolismo , Fatores Imunológicos/uso terapêutico , Neoplasias/tratamento farmacológico , Células A549 , Angiostrongylus cantonensis/genética , Animais , Apoptose/efeitos dos fármacos , Calreticulina/genética , Calreticulina/metabolismo , Calreticulina/farmacologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Células Hep G2 , Humanos , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Camundongos , Neoplasias/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
4.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 355-360, 2020 Aug 24.
Artigo em Chinês | MEDLINE | ID: mdl-32935508

RESUMO

OBJECTIVE: To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. METHODS: The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. RESULTS: SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. CONCLUSIONS: The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.


Assuntos
Proteínas de Helminto , Proteínas Recombinantes , Schistosoma japonicum , Animais , Anticorpos Anti-Helmínticos/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(32): 19299-19309, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32737161

RESUMO

Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease affecting over 200 million people. Schistosomes develop multiple body plans while navigating their complex life cycle, which involves two different hosts: a mammalian definitive host and a molluscan intermediate host. Their survival and propagation depend upon proliferation and differentiation of stem cells necessary for parasite homeostasis and reproduction. Infective larvae released from snails carry a handful of stem cells that serve as the likely source of new tissues as the parasite adapts to life inside the mammalian host; however, the role of these stem cells during this critical life cycle stage remains unclear. Here, we characterize stem cell fates during early intramammalian development. Surprisingly, we find that the esophageal gland, an accessory organ of the digestive tract, develops before the rest of the digestive system is formed and blood feeding is initiated, suggesting a role in processes beyond nutrient uptake. To explore such a role, we examine schistosomes that lack the esophageal gland due to knockdown of a forkhead-box transcription factor, Sm-foxA, which blocks development and maintenance of the esophageal gland, without affecting the development of other somatic tissues. Intriguingly, schistosomes lacking the esophageal gland die after transplantation into naive mice, but survive in immunodeficient mice lacking B cells. We show that parasites lacking the esophageal gland are unable to lyse ingested immune cells within the esophagus before passing them into the gut. These results unveil an immune-evasion mechanism mediated by the esophageal gland, which is essential for schistosome survival and pathogenesis.


Assuntos
Esôfago/parasitologia , Evasão da Resposta Imune , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Animais , Esôfago/imunologia , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Masculino , Camundongos , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/fisiopatologia
6.
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
8.
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
9.
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
10.
Parasitol Res ; 119(8): 2695-2702, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32556538

RESUMO

Opisthorchis felineus is a trematode flatworm that parasitises mammals, including humans, and is mainly spread throughout Eastern Europe and Western Siberia. The main drug used in treatment of opisthorchiasis and other trematode and cestode infestations is praziquantel (PZQ). We provide a possible explanation of PZQ-mediated tegument disruption. The idea is that the nature of tegument disruption is related to failure of surface renovation due to insufficiency of microtubule transport of vesicles. This insufficiency arises from microtubule destabilisation, which in the medium term leads to the decrease in tubulins alpha, beta and dynein mRNA amounts and deficiency of the corresponding proteins. We also found the upregulation of cGMP-dependent protein kinase gene, and we concluded that its protein product helped to overcome the effect of praziquantel and might be a promising target for combined anthelmintic therapy with PZQ. We concluded that function of saposin-like protein 2 (SAP2) is unlikely associated with membrane fusion, and SAP2 is probably able to bind some type of hydrophobic compounds including praziquantel.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/genética , Opisthorchis/efeitos dos fármacos , Praziquantel/farmacologia , Animais , Antiplatelmínticos/farmacologia , Antiplatelmínticos/uso terapêutico , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Opistorquíase/tratamento farmacológico , Praziquantel/uso terapêutico
11.
PLoS Negl Trop Dis ; 14(5): e0008242, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32401754

RESUMO

Alveolar and cystic echinococcosis (AE, CE) caused by E. multilocularis and E. granulosus s.l., respectively, are considered emerging zoonotic diseases in Kyrgyzstan with some of the world highest regional incidences. Little is known regarding the molecular variability of both species in Kyrgyzstan. In this study we provide molecular data from a total of 72 parasite isolates derived from humans (52 AE and 20 CE patients) and 43 samples from dogs (23 infected with E. multilocularis and 20 with E. granulosus s.l.).Genetic variability in E. multilocularis was studied using the concatenated complete sequences of the cob, nad2 and cox1 mitochondrial genes adding a total of 3,558bp per isolate. The cob/nad2/cox1 A2 haplotype was identified in 63.4% of the human and in 65.2% of the dog samples. This haplotype was originally described in samples from Kazakhstan and St. Lawrence Island (Alaska, USA). We also describe here 16 non-previously defined variants of E. multilocularis (called A11-A26). All haplotypes cluster together within the Asian group in the haplotype network. Based on Fst values, low level of genetic differentiation was found between the populations of E. multilocularis isolated from different regions within the country. However, high degree of differentiation was found when all the concatenated sequences from Kyrgyzstan are considered as a single population and compared with the population of the parasite from the neighbouring country China. In the case of E. granulosus s.l. the analysis was based in 1,609bp of the cox1 gene. One isolate from a dog was identified as E. equinus, while all the other sequences were identified belonging to E. granulosus s.s. In total, 24 cox1 haplotypes of E. granulosus s.s. were identified including the already described variants: Eg01 (in 6 samples), Eg33 (in 4 samples), EgCl04 (in 2 samples), Eg03 (in 1 sample) and Eg32 (in 1 sample). From the twenty-five other isolates of E. granulosus s.s. a total of 19 non-previously described cox1 haplotypes were identified and named as EgKyr1 to EgKyr19. The most common haplotype infecting human is the EgKyr1 which was found in 5 isolates.The cob/nad2/cox1 A2 haplotype of E. multilocularis is responsible for the majority of human infections in Kyrgyzstan and is also found in the majority of dogs included in this study. Further similar studies in different parts of Asia could elucidate if it is also the most common variant infecting humans in other countries. It remains unknown if this particular haplotype presents differences in virulence which could have contributed to the emergency of alveolar echinococcosis in Kyrgyzstan. In the case of E. granulosus s.s. it seems that there is no dominant haplotype infecting humans in Kyrgzstan. Further characterization of biological or antigenic features of dominant mitochondrial haplotypes could help to elucidate if they present differences which could be relevant in the diagnostic, pathogenicity or in the host/parasite interaction when infecting humans.


Assuntos
Doenças Transmissíveis Emergentes/parasitologia , Equinococose/parasitologia , Echinococcus granulosus/classificação , Echinococcus granulosus/genética , Echinococcus multilocularis/classificação , Echinococcus multilocularis/genética , Variação Genética , Adulto , Animais , Análise por Conglomerados , Doenças Transmissíveis Emergentes/epidemiologia , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Equinococose/epidemiologia , Equinococose/veterinária , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Haplótipos , Proteínas de Helminto/genética , Humanos , Incidência , Quirguistão/epidemiologia , Masculino , Tipagem de Sequências Multilocus , NADH Desidrogenase/genética
12.
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
13.
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
14.
Parasit Vectors ; 13(1): 154, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228657

RESUMO

BACKGROUND: Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host-parasite interaction has not been addressed yet. RESULTS: An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-ß1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. CONCLUSIONS: Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.


Assuntos
Cabras/parasitologia , Haemonchus/enzimologia , Haemonchus/genética , Leucócitos Mononucleares/imunologia , Metiltransferases/genética , Metiltransferases/imunologia , Metiltransferases/isolamento & purificação , Animais , Anticorpos Anti-Helmínticos/sangue , Proliferação de Células , Clonagem Molecular , Citocinas/metabolismo , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Regulação da Expressão Gênica , Hemoncose/parasitologia , Hemoncose/veterinária , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/imunologia , Masculino , Metiltransferases/metabolismo , Óxido Nítrico/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência de Proteína
15.
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
16.
Parasit Vectors ; 13(1): 164, 2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245505

RESUMO

BACKGROUND: Smad proteins are essential cellular mediators within the transforming growth factor-ß (TGF-ß) superfamily. They directly transmit incoming signals from the cell surface receptors to the nucleus. In spite of their functional importance, almost nothing is known about Smad proteins in parasitic nematodes including Haemonchus contortus, an important blood-sucking nematode of small ruminants. METHODS: Based on genomic and transcriptome data for H. contortus and using bioinformatics methods, a Smad homologue (called Hco-daf-8) was inferred from H. contortus and the structural characteristics of this gene and its encoded protein Hco-DAF-8 established. Using real-time PCR and immunofluorescence assays, temporal transcriptional and spatial expression profiles of Hco-daf-8 were studied. Gene rescue in Caenorhabditis elegans was then applied to assess the function of Hco-daf-8 and a specific inhibitor of human Smad3 (called SIS3) was employed to evaluate the roles of Hco-DAF-8 in H. contortus development. RESULTS: The features of Hco-DAF-8 (502 amino acids), including conserved R-Smad domains and residues of the L3-loop that determine pathway specificity, are consistent with a TGF-ß type I receptor-activated R-Smad. The Hco-daf-8 gene was transcribed in all developmental stages of H. contortus studied, with a higher level of transcription in the fourth-stage larval (L4) females and the highest level in adult males. Hco-DAF-8 was expressed in the platymyarian muscular cells, intestine and reproductive system of adult stages. Gene rescue experiments showed that Hco-daf-8 was able to partially rescue gene function in a daf-8 deficient mutant strain of C. elegans, leading to a resumption of normal development. In H. contortus, SIS3 was shown to affect H. contortus development from the exsheathed third-stage larvae (L3s) to L4s in vitro. CONCLUSIONS: These findings suggest that Hco-DAF-8, encoded by the gene Hco-daf-8, is an important cellular mediator of H. contortus development via the TGF-ß signalling pathway. They provide a basis for future explorations of Hco-DAF-8 and associated pathways in H. contortus and other important parasitic nematodes.


Assuntos
Haemonchus/genética , Proteínas de Helminto/genética , Proteínas Smad Reguladas por Receptor/genética , Transcriptoma , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Genômica , Haemonchus/crescimento & desenvolvimento , Masculino , Alinhamento de Sequência , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/classificação
17.
Parasit Vectors ; 13(1): 162, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238181

RESUMO

BACKGROUND: While immune responses to the murine hookworm Nippostrongylus brasiliensis have been investigated, signaling pathways regulating development of infectious larvae (iL3) are not well understood. We hypothesized that N. brasiliensis would use pathways similar to those controlling dauer development in the free-living nematode Caenorhabditis elegans, which is formally known as the "dauer hypothesis." METHODS: To investigate whether dafachronic acid activates the N. brasiliensis DAF-12 homolog, we utilized an in vitro reporter assay. We then utilized RNA-Seq and subsequent bioinformatic analyses to identify N. brasiliensis dauer pathway homologs and examine regulation of these genes during iL3 activation. RESULTS: In this study, we demonstrated that dafachronic acid activates the N. brasiliensis DAF-12 homolog. We then identified N. brasiliensis homologs for members in each of the four canonical dauer pathways and examined their regulation during iL3 activation by either temperature or dafachronic acid. Similar to C. elegans, we found that transcripts encoding antagonistic insulin-like peptides were significantly downregulated during iL3 activation, and that a transcript encoding a phylogenetic homolog of DAF-9 increased during iL3 activation, suggesting that both increased insulin-like and DAF-12 nuclear hormone receptor signaling accompanies iL3 activation. In contrast to C. elegans, we observed a significant decrease in transcripts encoding the dauer transforming growth factor beta ligand DAF-7 during iL3 activation, suggesting a different role for this pathway in parasitic nematode development. CONCLUSIONS: Our data suggest that canonical dauer pathways indeed regulate iL3 activation in the hookworm N. brasiliensis and that DAF-12 may be a therapeutic target in hookworm infections.


Assuntos
Colestenos/farmacologia , Nippostrongylus/efeitos dos fármacos , Nippostrongylus/genética , Transdução de Sinais/efeitos dos fármacos , Temperatura , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Filogenia , RNA-Seq
18.
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
19.
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
20.
Parasit Vectors ; 13(1): 151, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32204731

RESUMO

BACKGROUND: RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized. METHODS: Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat. RESULTS: rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. CONCLUSIONS: Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.


Assuntos
Hemoncose/parasitologia , Haemonchus/enzimologia , Haemonchus/genética , Metaloproteinase 12 da Matriz/genética , RNA Interferente Pequeno , Animais , Proliferação de Células , China , Feminino , Técnicas de Silenciamento de Genes , Cabras/parasitologia , Haemonchus/patogenicidade , Proteínas de Helminto/genética , Leucócitos Mononucleares , Masculino , Óxido Nítrico/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Análise de Sequência
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