scholarly journals Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion

2020 ◽  
Author(s):  
Cong Li ◽  
Qiping Zhao ◽  
Shunhai Zhu ◽  
Qingjie Wang ◽  
Haixia Wang ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Apical Membrane ◽  
Specific Protein ◽  
Host Cells ◽  
Host Cell Invasion ◽  
Secreted Protein ◽  
Interacting Proteins ◽  
Apical Membrane Antigen 1

Abstract Background: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several species of the protozoan parasite Eimeria. Among these species, E. tenella causes hemorrhagic pathologies and high mortality. These parasites have complex and diverse lifestyles that require the invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp).Methods: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C.The localization of EtEsp was analyzed with an immunofluorescence assay. An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites.Results: The interaction between EtAMA1 and EtEsp was confirmed with BiFC in vivo and by GST pull-down in vitro. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life cycle. An immunofluorescence analysis showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells in vitro. Conclusion: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in the sporozoite invasion of host cells and maybe involved in the growth of the parasite in the host. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

scholarly journals Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion

2020 ◽  
Author(s):  
Cong Li ◽  
Qiping Zhao ◽  
Shunhai Zhu ◽  
Qingjie Wang ◽  
Haixia Wang ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Apical Membrane ◽  
Specific Protein ◽  
Host Cells ◽  
Host Cell Invasion ◽  
Secreted Protein ◽  
Interacting Proteins ◽  
Apical Membrane Antigen 1

Abstract Background: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several species of the protozoan parasite Eimeria. Among these species, E. tenella causes hemorrhagic pathologies and high mortality. These parasites have complex and diverse lifestyles that require the invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp).Methods: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C.The localization of EtEsp was analyzed with an immunofluorescence assay. An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites.Results: The interaction between EtAMA1 and EtEsp was confirmed with BiFC in vivo and by GST pull-down in vitro. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life cycle. An immunofluorescence analysis showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells in vitro. Conclusion: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in the sporozoite invasion of host cells and maybe involved in the growth of the parasite in the host. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

scholarly journals Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion

2019 ◽  
Author(s):  
Cong Li ◽  
Qiping Zhao ◽  
Shunhai Zhu ◽  
Qingjie Wang ◽  
Haixia Wang ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Apical Membrane ◽  
Specific Protein ◽  
Host Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Host Cell Invasion ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

Abstract Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In this study, we characterized one putative Et AMA1-interacting protein, E. tenella Eimeria -specific protein ( Et Esp). The interaction between Et AMA1 and Et Esp was confirmed with bimolecular fluorescence complementation (BiFC) in vivo and by glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) in vitro . We showed that Et Esp is differentially expressed during distinct phases of the parasite life cycle by using qPCR and western blotting. Immunofluorescence analysis showed that the Et Esp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that Et Esp is a micronemal protein secreted by sporozoites. In inhibition tests, a polyclonal anti-r Et Esp antibody attenuated the capacity of E. tenella to invade host cells in vitro . These data have implications for the use of Et AMA1 or Et AMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

scholarly journals 1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1053
Author(s):  
Lidia Węglińska ◽  
Adrian Bekier ◽  
Katarzyna Dzitko ◽  
Barbara Pacholczyk-Sienicka ◽  
Łukasz Albrecht ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Direct Action ◽  
Human Fibroblasts ◽  
Imidazole Ring ◽  
Host Cells ◽  
In Vitro Assays ◽  
Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

scholarly journals Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Jennifer Timm ◽  
Maria Valente ◽  
Daniel García-Caballero ◽  
Keith S. Wilson ◽  
Dolores González-Pacanowska
Keyword(s):  
Homologous Recombination ◽  
Host Cell ◽  
Trypanosoma Brucei ◽  
Cell Invasion ◽  
Essential Amino Acids ◽  
Host Cell Invasion ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACT Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction. Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

scholarly journals Conditional Expression of Toxoplasma gondii Apical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

2005 ◽  
Vol 16 (9) ◽  
pp. 4341-4349 ◽  
Author(s):  
Jeffrey Mital ◽  
Markus Meissner ◽  
Dominique Soldati ◽  
Gary E. Ward
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Apical Membrane ◽  
Conditional Knockout ◽  
Host Cell Invasion ◽  
Apical Membrane Antigen 1 ◽  
Apicomplexan Parasites ◽  
Apical Membrane Antigen ◽  
Conditional Expression

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.

scholarly journals Independent Roles of Apical Membrane Antigen 1 and Rhoptry Neck Proteins during Host Cell Invasion by Apicomplexa

2011 ◽  
Vol 10 (6) ◽  
pp. 591-602 ◽  
Author(s):  
Donatella Giovannini ◽  
Stephan Späth ◽  
Céline Lacroix ◽  
Audrey Perazzi ◽  
Daniel Bargieri ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Apical Membrane ◽  
Host Cell Invasion ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

scholarly journals AMA1-Deficient Toxoplasma gondii Parasites Transiently Colonize Mice and Trigger an Innate Immune Response That Leads to Long-Lasting Protective Immunity

2015 ◽  
Vol 83 (6) ◽  
pp. 2475-2486 ◽  
Author(s):  
Vanessa Lagal ◽  
Márcia Dinis ◽  
Dominique Cannella ◽  
Daniel Bargieri ◽  
Virginie Gonzalez ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Distinct Type ◽  
Genetically Engineered ◽  
Host Cells ◽  
Type I ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Immunocompromised Mice

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera,PlasmodiumandToxoplasma, until we genetically engineered viable parasites lackingAMA1. The reduction in invasiveness of theToxoplasma gondiiRH-AMA1 knockout (RH-AMA1KO) tachyzoite population,in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1KOtachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1KOpopulation amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type IIT. gondiigenotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite populationin vivo.

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