Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host

Sm16 is an immunomodulatory protein that seems to play a key role in the suppression of the cutaneous inflammatory response during Schistosoma mansoni penetration of the skin of definitive hosts. Therefore, Sm16 represents a potential target for protective immune responses induced by vaccination. In this work, we generated the recombinant protein rSm16 and produced polyclonal antibodies against this protein to evaluate its expression during different parasite life-cycle stages and its location on the surface of the parasite. In addition, we analyzed the immune responses elicited by immunization with rSm16 using two different vaccine formulations, as well as its ability to induce protection in Balb/c mice. In order to explore the biological function of Sm16 during the course of experimental infection, RNA interference was also employed. Our results demonstrated that Sm16 is expressed in cercaria and schistosomula and is located in the schistosomula surface. Despite humoral and cellular immune responses triggered by vaccination using rSm16 associated with either Freund’s or alum adjuvants, immunized mice presented no reduction in either parasite burden or parasite egg laying. Knockdown of Sm16 gene expression in schistosomula resulted in decreased parasite size in vitro but had no effect on parasite survival or egg production in vivo. Thus, our findings demonstrate that although the vaccine formulations used in this study succeeded in activating immune responses, these failed to promote parasite elimination. Finally, we have shown that Sm16 is not vital for parasite survival in the definitive host and hence may not represent a suitable target for vaccine development.

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Structure-Reactivity Relationships on Substrates and Inhibitors of the Lysine Deacylase Sirtuin 2 from Schistosoma Mansoni (SmSirt2)

10.26434/chemrxiv.7957544 ◽

2019 ◽

Cited By ~ 1

Author(s):

Daria Monaldi ◽

Dante Rotili ◽

Julien Lancelot ◽

Martin Marek ◽

Nathalie Wössner ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Human Cancer ◽

Egg Laying ◽

Human Cancer Cells ◽

Parasite Survival ◽

Survival And Reproduction ◽

Emergence Of Resistance ◽

First Time ◽

Parasitic Life Cycle

The only drug for treatment of Schistosomiasis is Praziquantel, and the possible emergence of resistance makes research on novel therapeutic agents necessary. Targeting of Schistosoma mansoni epigenetic enzymes, which regulate the parasitic life cycle, emerged as promising approach. Due to the strong effects of human Sirtuin inhibitors on parasite survival and reproduction, Schistosoma sirtuins were postulated as therapeutic targets. In vitro testing of synthetic substrates of S. mansoni Sirtuin 2 (SmSirt2) and kinetic experiments on a myristoylated peptide demonstrated lysine long chain deacylation as an intrinsic SmSirt2 activity for the first time. Focused in vitro screening of the GSK Kinetobox library and structure-activity relationships (SAR) of identified hits, led to the first SmSirt2 inhibitors with activity in the low micromolar range. Several SmSirt2 inhibitors showed potency against both larval schistosomes (viability) and adult worms (pairing, egg laying) in culture without general toxicity to human cancer cells.

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Protective Immunity Against Neospora Caninum Infection Induced by 14-3-3 Protein in Mice

10.21203/rs.3.rs-114643/v1 ◽

2020 ◽

Author(s):

Shan Li ◽

Nan Zhang ◽

Shaoxiong Liu ◽

Jianhua Li ◽

Li Liu ◽

...

Keyword(s):

Immune Responses ◽

Vaccine Development ◽

Neospora Caninum ◽

Survival Rates ◽

Parasite Burden ◽

Mouse Serum ◽

Intermediate Hosts ◽

Caninum Infection ◽

Wide Range ◽

Ifn Γ

Abstract BackgroundNeospora caninum causes infections in a wide range of intermediate hosts and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Our previous studies demonstrated that N. caninum 14-3-3 protein (Nc14-3-3), which is included in N. caninum extracellular vesicles (NEVs), can induce effective immune responses and stimulate cytokine expression in mouse peritoneal macrophages. However, whether Nc14-3-3 has a protective effect and its mechanisms are poorly understood.MethodsHere, we evaluated immune responses and protective effects of Nc14-3-3 against 2×107 Nc-1 tachyzoites. Antibody (IgG, IgGl and IgG2a) levels and Th1-type (IFN-γ and IL-12) and Th2-type (IL-4 and IL-10) cytokines in mouse serum; survival rates; survival time; and parasite burdens were detected.ResultsIn the present study, the immunostimulatory effect of Nc14-3-3 was confirmed, as it triggered Th1-type cytokine (IFN-γ and IL-12) production in mouse serum two weeks after the final immunization. Moreover, the immunization of C57BL/6 mice with Nc14-3-3 induced high IgG antibody levels and significant increases in CD8+ T lymphocytes in the spleens of mice, indicating that a significant cellular immune response was induced. Mouse survival rates and survival times were significantly prolonged after immunization survival rates were 40% for Nc14-3-3 immunization and 60% for NEV immunization, while mice that received GST, PBS, or blank control all died at 13, 9, and 8 days after intraperitoneal N. caninum challenge. In addition, qPCR analysis indicated that there was a lower parasite burden and milder pathological changes in the mice immunized with Nc14-3-3.ConclusionsOur data demonstrate the vaccination of mice with Nc14-3-3 elicits both cellular and humoural immune responses and provides partial protection against acute neosporosis. Thus, Nc14-3-3 could be an effective antigen candidate for vaccine development for neosporosis.

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Protective Immunity Against Neospora caninum Infection Induced by 14-3-3 Protein in Mice

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.638173 ◽

2021 ◽

Vol 8 ◽

Author(s):

Shan Li ◽

Nan Zhang ◽

Shaoxiong Liu ◽

Jianhua Li ◽

Li Liu ◽

...

Keyword(s):

Immune Responses ◽

Vaccine Development ◽

Neospora Caninum ◽

Survival Rates ◽

Parasite Burden ◽

Mouse Serum ◽

Protective Effects ◽

Igg Antibody ◽

Caninum Infection ◽

Ifn Γ

Neospora caninum is an apicomplexan parasite that infects many mammals and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Our previous studies demonstrated that N. caninum 14-3-3 protein (Nc14-3-3), which is included in N. caninum extracellular vesicles (NEVs), can induce effective immune responses and stimulate cytokine expression in mouse peritoneal macrophages. However, whether Nc14-3-3 has a protective effect and its mechanisms are poorly understood. Here, we evaluated the immune responses and protective effects of Nc14-3-3 against exposure to 2 × 107 Nc-1 tachyzoites. Antibody (IgG, IgGl, and IgG2a) levels and Th1-type (IFN-γ and IL-12) and Th2-type (IL-4 and IL-10) cytokines in mouse serum, survival rates, survival times, and parasite burdens were detected. In the present study, the immunostimulatory effect of Nc14-3-3 was confirmed, as it triggered Th1-type cytokine (IFN-γ and IL-12) production in mouse serum 2 weeks after the final immunization. Moreover, the immunization of C57BL/6 mice with Nc14-3-3 induced high IgG antibody levels and significant increases in CD8+ T lymphocytes in the spleens of mice, indicating that the cellular immune response was significantly stimulated. Mouse survival rates and times were significantly prolonged after immunization; the survival rates were 40% for Nc14-3-3 immunization and 60% for NEV immunization, while mice that received GST, PBS, or blank control all died at 13, 9, or 8 days, respectively, after intraperitoneal N. caninum challenge. In addition, qPCR analysis indicated that there was a reduced parasite burden and diminished pathological changes in the mice immunized with Nc14-3-3. Our data demonstrate that vaccination of mice with Nc14-3-3 elicits both cellular and humoral immune responses and provides partial protection against acute neosporosis. Thus, Nc14-3-3 could be an effective antigen candidate for vaccine development for neosporosis.

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Vaccination with Schistosoma mansoni cholinesterases reduces parasite burden and egg viability in a mouse model of schistosomiasis

10.1101/2020.01.28.922732 ◽

2020 ◽

Author(s):

Bemnet A. Tedla ◽

Darren Pickering ◽

Luke Becker ◽

Alex Loukas ◽

Mark S. Pearson

Keyword(s):

Schistosoma Mansoni ◽

Parasite Burden ◽

Egg Viability ◽

Egg Hatching ◽

Parasite Survival ◽

Liver Homogenates ◽

Secretory Products ◽

Glycogen Stores ◽

Independent Trials

AbstractSchistosomiasis is a neglected tropical disease which kills 300,000 people every year in developing countries and there is no vaccine. Recently, we have shown that cholinesterases (ChEs) - enzymes that regulate neurotransmission - from Schistosoma mansoni are expressed on the tegument and present in the excretory/secretory products of schistosomula and adult worms, and are essential for parasite survival in the definitive host, highlighting their utility as potential schistosomiasis vaccine targets. When treated in vitro with anti-SmChE IgG, both schistosomula and adult worms displayed significantly decreased ChE activity, which eventually resulted in parasite death. Vaccination with individual SmChEs, or a combination of all three SmChEs, significantly reduced worm burdens in two independent trials compared to controls. Liver egg burdens were significantly decreased for all vaccinated mice across both trials (13% - 46%) except for those vaccinated with SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE2 (46%). Further, surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. These results suggest that SmChEs could be incorporated into a vaccine against schistosomiasis to reduce the pathology and transmission of this debilitating disease.

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Structure-Reactivity Relationships on Substrates and Inhibitors of the Lysine Deacylase Sirtuin 2 from Schistosoma Mansoni (SmSirt2)

10.26434/chemrxiv.7957544.v1 ◽

2019 ◽

Author(s):

Daria Monaldi ◽

Dante Rotili ◽

Julien Lancelot ◽

Martin Marek ◽

Alessia Lucidi ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Human Cancer ◽

Egg Laying ◽

Standard Drug ◽

Human Cancer Cells ◽

Parasite Survival ◽

Survival And Reproduction ◽

Emergence Of Resistance ◽

Parasitic Life Cycle

The standard drug for treatment of the neglected disease Schistosomiasis is Praziquantel, and the possible emergence of resistance to this treatment makes the research on novel therapeutic agents necessary and urgent. To this end, the targeting of Schistosoma mansoni epigenetic enzymes, which regulate the parasitic life cycle, emerged as promising approach. Due to strong effects of human Sirtuin inhibitors on parasite survival and reproduction, Schistosoma sirtuins were postulated as potential therapeutic targets. In vitro testing of synthetic substrates of SmSirt2 and kinetic experiments on a myristoylated peptide newly demonstrated lysine long chain deacylation as an intrinsic SmSirt2 activity in addition to the known deacetylation. Focused in vitro screening of the GSK Kinetobox library and structure-activity relationships (SAR) of identified hits, led to the first SmSirt2 inhibitors with activity in the low micromolar range. Several SmSirt2 inhibitors showed potency against both larval schistosomes (viability) and adult worms (pairing, egg laying) in culture without general toxicity to human cancer cells.

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Vaccination with Schistosoma mansoni Cholinesterases Reduces the Parasite Burden and Egg Viability in a Mouse Model of Schistosomiasis

Vaccines ◽

10.3390/vaccines8020162 ◽

2020 ◽

Vol 8 (2) ◽

pp. 162 ◽

Cited By ~ 3

Author(s):

Bemnet A. Tedla ◽

Darren Pickering ◽

Luke Becker ◽

Alex Loukas ◽

Mark S. Pearson

Keyword(s):

Schistosoma Mansoni ◽

Parasite Burden ◽

Egg Viability ◽

Egg Hatching ◽

Blood Flukes ◽

Parasite Survival ◽

Liver Homogenates ◽

Secretory Products ◽

Glycogen Stores

Schistosomiasis is a neglected tropical disease caused by parasitic blood flukes of the genus Schistosoma, which kills 300,000 people every year in developing countries, and there is no vaccine. Recently, we have shown that cholinesterases (ChEs)—enzymes that regulate neurotransmission—from Schistosoma mansoni are expressed on the outer tegument surface and present in the excretory/secretory products of larval schistosomula and adult worms, and are essential for parasite survival in the definitive host, highlighting their utility as potential schistosomiasis vaccine targets. When treated in vitro with anti-schistosome cholinesterase (SmChE) IgG, both schistosomula and adult worms displayed significantly decreased ChE activity, which eventually resulted in parasite death. Vaccination with individual SmChEs, or a combination of all three SmChEs, significantly reduced worm burdens in two independent trials compared to controls. Average adult worm numbers and liver egg burdens were significantly decreased for all vaccinated mice across both trials, with values of 29–39% and 13–46%, respectively, except for those vaccinated with SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE2 (46%). Furthermore, surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. These results suggest that SmChEs could be incorporated into a vaccine against schistosomiasis to reduce the pathology and transmission of this debilitating disease.

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Ameliorative effects of Schisandrin B on Schistosoma mansoni-induced hepatic fibrosis in vivo

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009554 ◽

2021 ◽

Vol 15 (6) ◽

pp. e0009554

Author(s):

Ho Yin Pekkle Lam ◽

Ting-Ruei Liang ◽

Shih-Yi Peng

Keyword(s):

Liver Fibrosis ◽

Schistosoma Mansoni ◽

Egg Production ◽

Treatment Options ◽

Parasite Burden ◽

Inflammasome Activation ◽

Schisandrin B ◽

Male Adult

Schistosomiasis is second only to malaria as the most devastating parasitic disease in the world. It is caused by the helminths Schistosoma mansoni (S. mansoni), S. haematobium, or S. japonicum. Typically, patients with schistosomiasis suffer from symptoms of liver fibrosis and hepatosplenomegaly. Currently, patients were treated with praziquantel. Although praziquantel effectively kills the worm, it cannot prevent re-infection or resolve liver fibrosis. Also, current treatment options are not ample to completely cure liver fibrosis and splenic damages. Moreover, resistance of praziquantel has been reported in vivo and in vitro studies. Therefore, finding new effective treatment agents is urgently needed. Schisandrin B (Sch B) of Schisandra chinensis has been shown to protect against different liver injuries including fatty liver disease, hepatotoxicity, fibrosis, and hepatoma. We herein investigate the potential of using Sch B to treat S. mansoni-induced liver fibrosis. Results from the present study demonstrate that Sch B is beneficial in treating S. mansoni-induced liver fibrosis and splenic damages, through inhibition of inflammasome activation and apoptosis; and aside from that regulates host immune responses. Besides, Sch B treatment damages male adult worm in the mice, consequently helps to reduce egg production and lessen the parasite burden.

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Structure-Reactivity Relationships on Substrates and Inhibitors of the Lysine Deacylase Sirtuin 2 from Schistosoma Mansoni (SmSirt2)

10.26434/chemrxiv.7957544.v2 ◽

2019 ◽

Author(s):

Daria Monaldi ◽

Dante Rotili ◽

Julien Lancelot ◽

Martin Marek ◽

Nathalie Wössner ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Human Cancer ◽

Egg Laying ◽

Human Cancer Cells ◽

Parasite Survival ◽

Survival And Reproduction ◽

Emergence Of Resistance ◽

First Time ◽

Parasitic Life Cycle

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Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) induces global transcriptional deregulation and ultrastructural alterations that impair viability in Schistosoma mansoni

10.1101/829549 ◽

2019 ◽

Author(s):

Vitor Coutinho Carneiro ◽

Isabel Caetano de Abreu da Silva ◽

Murilo Sena Amaral ◽

Adriana S.A. Pereira ◽

Gilbert O. Silveira ◽

...

Keyword(s):

Gene Expression ◽

Schistosoma Mansoni ◽

Drug Target ◽

Egg Production ◽

Egg Laying ◽

New Drugs ◽

Biological Processes ◽

Lysine Specific Demethylase

AbstractTreatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ), and due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935. We synthesized a novel and potent LSD1 inhibitor, MC3935, which was used to treat schistosomula or adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.Author SummarySchistosomiasis mansoni is a chronic and debilitating tropical disease caused by the helminth Schistosoma mansoni. The control and treatment of the disease rely almost exclusively on praziquantel (PZQ). Thus, there is an urgent need to search for promising protein targets to develop new drugs. Drugs that inhibit enzymes that modify the chromatin structure have been developed for a number of diseases. We and others have shown that S. mansoni epigenetic enzymes are also potential therapeutic targets. Here we evaluated the potential of the S. mansoni histone demethylase LSD1 (SmLSD1) as a drug target. We reported the synthesis of a novel and potent LSD1 inhibitor, MC3935, and show that it selectively inhibited the enzymatic activity of SmLSD1. Treatment of juvenile or adult worms with MC3935 caused severe damage to the tegument of the parasites and compromised egg production. Importantly, MC3935 proved to be highly toxic to S. mansoni, culminating in the death of juvenile or adult worms within 96 h. Transcriptomic analysis of MC3935-treated parasites revealed changes in the gene expression of hundreds of genes involved in key biological processes. Importantly, SmLSD1 contains unique sequences within its polypeptide chain that could be explored to develop a S. mansoni selective drug.

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Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) is a druggable target involved in parasite survival, oviposition and stem cell proliferation

10.1101/2020.09.17.301184 ◽

2020 ◽

Author(s):

G. Padalino ◽

C. A. Celatka ◽

H. Y. Rienhoff ◽

J. H. Kalin ◽

P. A. Cole ◽

...

Keyword(s):

Cell Proliferation ◽

Stem Cell ◽

Schistosoma Mansoni ◽

Adult Worm ◽

Histone Methylation ◽

Egg Production ◽

Regulation Of Gene Expression ◽

Stem Cell Proliferation ◽

Lysine Specific Demethylase ◽

Parasite Survival

AbstractSchistosomiasis is a chronically-debilitating neglected tropical disease (NTD) that predominantly affects people living in resource-poor communities of tropical and subtropical countries. Schistosoma mansoni, one of three species responsible for most human infections, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic- and epigenetic- processes. As inhibition of S. mansoni epigenetic machinery components has been shown to impair key transitions throughout the parasite’s digenetic lifecycle, this knowledge is currently fuelling the search for new epi-drug - based anthelmintics.In this study, the anti-schistosomal activity of 39 re-purposed Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors was investigated on key life cycle stages associated with both definitive (schistosomula, juvenile worms, sexually-mature adults) and intermediate host (miracidia) infection. The most active compound (compound 33; e.g. schistosomula phenotype EC50 = 4.370 µM; adult worm motility EC50 = 2.137 µM) was subsequently used to provide further insight into the critical role of S. mansoni lysine specific demethylase 1 (SmLSD1) in adult worm oviposition and stem cell proliferation. Here, compound 33 treatment of adult schistosomes led to significant defects in egg production, intra-egg vitellocyte/ovum packaging and gonadal/neoblast stem cell proliferation. A greater abundance of H3K4me2 marks accompanied these phenotypes and supported specific inhibition of SmLSD1 in adult schistosomes by compound 33. In silico screening indicated that compound 33 likely inhibits SmLSD1 activity by covalently reacting with the FAD cofactor.This work suggests that evaluation of HsLSD1 - targeting epi-drugs could have utility in the search for next-generation anti-schistosomals. The ability of compound 33 to inhibit parasite survival, oviposition, H3K4me2 demethylation and stem cell proliferation warrants further investigations of this compound and its epigenetic target. This data further highlights the importance of histone methylation in S. mansoni lifecycle transitions.Author summaryAffecting over 200 million people, schistosomiasis is a chronic disease caused by the parasitic worm Schistosoma mansoni. The frontline drug for schistosomiasis treatment is praziquantel. Owing to the concern surrounding praziquantel insensitivity or resistance developing, current research is directed towards the identification of novel drugs. We have focused our search for compounds that affect essential aspects of schistosome biology including parasite movement, fertility, cell proliferation and survival. Since all of these functions are potentially influenced by epigenetic regulation of gene expression, we investigated the activity of compounds that alter histone methylation status. In this report, we show that S. mansoni Lysine Specific Demethylase 1 (SmLSD1), a histone demethylase, is critical to miracidia-to-sporocyst transitioning, adult worm motility, egg production and parasite survival. Inhibition of SmLSD1 with compounds developed to inhibit the human paralog show promising potential as novel anti-schistosomal agents.

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