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Yue Y, Zhang Y, Cheng Y, Jiao H, Yan M. Echinococcus granulosus antigen B regulates T-cell function through inhibition of signal transducer and activator of transcription 3 in experimental immune thrombocytopenia. Br J Haematol 2025. [PMID: 40325612 DOI: 10.1111/bjh.20064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2024] [Accepted: 03/21/2025] [Indexed: 05/07/2025]
Abstract
Dysregulated T-cell homeostasis is central to the development of immune thrombocytopenia (ITP), characterized by reduced platelet counts. Antigen B (AgB), a key protein in Echinococcus granulosus cyst fluid, modulates T-cell differentiation and reduces inflammation. Here, we explored the role of AgB in ITP and found that it enhances the generation and function of regulatory T cells (Tregs), boosting their immunosuppressive activity. In our passive ITP murine model, AgB treatment alleviated thrombocytopenia and restored the Treg-helper T-cell (Th) balance. However, the therapeutic effects of AgB on CD4+ T cells were abolished by Treg depletion, highlighting the essential role of Tregs in AgB's mechanism of action. Moreover, AgB reduced proinflammatory cytokine production and inhibited signal transducer and activator of transcription 3 (STAT3) activation in ITP mice, with STAT3 inhibition negating the effects of AgB in Tregs. AgB promoted STAT3 degradation via tumour necrosis factor receptor-associated factor 6 (TRAF6)-mediated ubiquitination. In conclusion, by facilitating TRAF6-mediated STAT3 ubiquitination, AgB restores T-cell homeostasis and strengthens Treg immunosuppression, affording a potential therapeutic strategy for ITP.
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Affiliation(s)
- Yingbin Yue
- The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Medical University, Urumqi, China
| | - Yunfei Zhang
- The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China
| | - Yongfeng Cheng
- The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China
| | - Hongjie Jiao
- The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China
| | - Mei Yan
- The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China
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Qian YY, Huang FF, Chen SY, Zhang WX, Wang Y, Du PF, Li G, Ding WB, Qian L, Zhan B, Chu L, Jiang DH, Yang XD, Zhou R. Therapeutic effect of recombinant Echinococcus granulosus antigen B subunit 2 protein on sepsis in a mouse model. Parasit Vectors 2024; 17:467. [PMID: 39548530 PMCID: PMC11566433 DOI: 10.1186/s13071-024-06540-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2024] [Accepted: 10/17/2024] [Indexed: 11/18/2024] Open
Abstract
BACKGROUND Sepsis is a potentially fatal systemic inflammatory response syndrome (SIRS) that threatens millions of lives worldwide. Echinococcus granulosus antigen B (EgAgB) is a protein released by the larvae of the tapeworm. This protein has been shown to play an important role in modulating host immune response. In this study we expressed EgAgB as soluble recombinant protein in E. coli (rEgAgB) and explored its protective effect on sepsis. METHODS The sepsis model was established by cecal ligation and puncture (CLP) procedure in BALB/c mice. The therapeutic effect of rEgAgB on sepsis was performed by interperitoneally injecting 5 µg rEgAgB in mice with CLP-induced sepsis and observing the 72 h survival rate after onset of sepsis. The proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6] and regulatory cytokines [IL-10, transforming growth factor beta (TGF-β)] were measured in sera, and the histopathological change was observed in livers, kidneys, and lungs of septic mice treated with rEgAgB compared with untreated mice. The effect of rEgAgB on the macrophage polarization was performed in vitro by incubating rEgAgB with peritoneal macrophages. The levels of TLR2 and MyD88 were measured in these tissues to determine the involvement of TLR-2/MyD88 in the sepsis-induced inflammatory signaling pathway. RESULTS In vivo, we observed that treatment with rEgAgB significantly increased the survival rate of mice with CLP-induced sepsis up to 72 h while all mice without treatment died within the same period. The increased survival was associated with reduced pathological damage in key organs such as liver, lung, and kidneys. It was supported by the reduced proinflammatory cytokine levels and increased regulatory cytokine expression in peripheral blood and key organ tissues. Further study identified that treatment with rEgAgB promoted macrophage polarization from classically activated macrophage (M1) to regulatory M2-like macrophage via inhibiting TLR2/MyD88 signal pathway. CONCLUSIONS The therapeutic effects of rEgAgB on mice with sepsis was observed in a mice model that was associated with reduced inflammatory responses and increased regulatory responses, possibly through inducing polarization of macrophages from proinflammatory M1 to regulatory M2 phenotype through inhibiting TLR2/MyD88 inflammatory pathway.
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Affiliation(s)
- Ya-Yun Qian
- First Affiliated Hospital of Bengbu Medical University, Bengbu, 233000, China
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
- First People's Hospital of Changzhou, Changzhou, 213000, China
| | - Fei-Fei Huang
- First Affiliated Hospital of Bengbu Medical University, Bengbu, 233000, China
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
| | - Si-Yu Chen
- Wuxi School of Medicine, Jiangnan University, Wuxi, 214028, China
- Department of Critical Care Medicine, Affiliated Hospital of Jiangnan University, Wuxi, 214122, China
| | - Wei-Xiao Zhang
- First Affiliated Hospital of Bengbu Medical University, Bengbu, 233000, China
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
| | - Yin Wang
- Department of Critical Care Medicine, Affiliated Hospital of Jiangnan University, Wuxi, 214122, China
| | - Peng-Fei Du
- Department of Critical Care Medicine, Affiliated Hospital of Jiangnan University, Wuxi, 214122, China
| | - Gen Li
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
| | - Wen-Bo Ding
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
| | - Lei Qian
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
| | - Bin Zhan
- National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Liang Chu
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China
- Second Affiliated Hospital of Bengbu Medical University, Bengbu, 233000, China
| | - Dong-Hui Jiang
- Department of Critical Care Medicine, Affiliated Hospital of Jiangnan University, Wuxi, 214122, China.
- Department of Critical Care Medicine, First People's Hospital of Haidong, Haidong, 810600, China.
| | - Xiao-Di Yang
- Anhui Key Laboratory of Infection and Immunity of Bengbu Medical University, Bengbu, 233000, China.
| | - Rui Zhou
- First Affiliated Hospital of Bengbu Medical University, Bengbu, 233000, China.
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Folle AM, Lagos Magallanes S, Fló M, Alvez-Rosado R, Carrión F, Vallejo C, Watson D, Julve J, González-Sapienza G, Pristch O, González-Techera A, Ferreira AM. Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation. Front Cell Infect Microbiol 2024; 14:1362765. [PMID: 38562963 PMCID: PMC10982386 DOI: 10.3389/fcimb.2024.1362765] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2023] [Accepted: 02/26/2024] [Indexed: 04/04/2024] Open
Abstract
Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1β, IL-6, IL-12p40, IFN-β) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.
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Affiliation(s)
- Ana Maite Folle
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
| | - Sofía Lagos Magallanes
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
| | - Martín Fló
- Unidad de Biofísica de Proteínas, Institut Pasteur, Montevideo, Uruguay
| | - Romina Alvez-Rosado
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
| | - Federico Carrión
- Unidad de Biofísica de Proteínas, Institut Pasteur, Montevideo, Uruguay
| | - Cecilia Vallejo
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
| | - David Watson
- Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom
| | - Josep Julve
- Research group of Endocrinology, Diabetes and Nutrition, Institut de Recerca SANT PAU, Barcelona, Spain
- Centro de Investigación Biomédica en red de Diabetes y Enfermedades Metabólicas asociadas, Instituto de Salud Carlos III, Madrid, Spain
| | - Gualberto González-Sapienza
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
| | - Otto Pristch
- Unidad de Biofísica de Proteínas, Institut Pasteur, Montevideo, Uruguay
| | - Andrés González-Techera
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
| | - Ana María Ferreira
- Unidad de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
- Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Montevideo, Uruguay
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Blanco V, Mozzo B, Alvite G. Dimerization, host-parasite communication and expression studies of an Echinococcus granulosus 2DBD nuclear receptor. Parasitol Res 2023; 122:2055-2063. [PMID: 37395819 DOI: 10.1007/s00436-023-07905-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2023] [Accepted: 06/16/2023] [Indexed: 07/04/2023]
Abstract
Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate various biological processes, such as metabolism, development and reproduction. Although NRs with two DNA-binding domains (2DBD) were identified in Schistosoma mansoni (Platyhelminth, Trematoda) more than fifteen years ago, these proteins have been poorly studied. 2DBD-NRs could become attractive therapeutic targets to combat parasitic diseases such as cystic echinococcosis since this type of protein is absent in vertebrate hosts. Cystic echinococcosis is a worldwide zoonosis caused by the larval stage of the parasitic platyhelminth Echinococcus granulosus (Cestoda) that generates an important public health problem and a significant economic loss. Recently, our research group identified four 2DBD-NRs in E. granulosus, named Eg2DBDα, Eg2DBDα.1 (an isoform of Eg2DBDα), Eg2DBDβ, and Eg2DBDγ. This work demonstrated that Eg2DBDα.1 forms homodimers through the E and F regions, whereas its interaction with EgRXRβa could not be detected. In addition, the stimulation of Eg2DBDα.1 homodimerization by intermediate host serum was shown, suggesting that at least one lipophilic molecule from bovine serum could bind to Eg2DBDα.1. Finally, Eg2DBDs expression studies in the protoscolex larval stage were performed, indicating that Eg2dbdγ is not expressed, whereas Eg2dbdα has the highest expression level followed by Eg2dbdβ and Eg2dbdα.1 in decreased order. Overall, these findings provide new insights into the mechanism of action of Eg2DBDα.1 and its potential role in host-parasite communication.
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Affiliation(s)
- Valentina Blanco
- Biochemistry Section, Faculty of Sciences, Universidad de la República, Montevideo, Uruguay
- Functional Genomics Laboratory, Instituto Pasteur de Montevideo, Montevideo, Uruguay
| | - Bruno Mozzo
- Biochemistry Section, Faculty of Sciences, Universidad de la República, Montevideo, Uruguay
| | - Gabriela Alvite
- Biochemistry Section, Faculty of Sciences, Universidad de la República, Montevideo, Uruguay.
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Mariki A, Barzin Z, Fasihi Harandi M, Karbasi Ravari K, Davoodi M, Mousavi SM, Rezakhani S, Nazeri M, Shabani M. Antigen B modulates anti-inflammatory cytokines in the EAE model of multiple sclerosis. Brain Behav 2023; 13:e2874. [PMID: 36582052 PMCID: PMC9927863 DOI: 10.1002/brb3.2874] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 12/13/2021] [Accepted: 01/22/2022] [Indexed: 12/31/2022] Open
Abstract
INTRODUCTION Multiple sclerosis (MS) is characterized by the destruction of the blood-brain barrier, loss of myelin sheath, and contribution of inflammatory interleukins such as TNF-alpha, interleukin-17, and interleukin-6. METHODS The current study investigated the effect of antigen B of hydatid cyst fluid on the reduction of anti-inflammatory cytokines and nerve conduction velocity in rats with experimental autoimmune encephalomyelitis (EAE)-induced MS. After isolation of antigen B from sterile cyst fluid, the rats were randomly divided into four groups: saline, EAE, EAE + teriflunomide (EAE + TF), and EAE + antigen B (EAE + AngB). The EAE model was induced using cow spinal cord homogenization, in combination with Freund's complete adjuvant. The serum concentration of cytokines including IL-1B and IL-17, IL-10, IL-6, and TNF-X was measured by the ELISA method, and real-time PCR was performed to study gene expression. Electrophysiological, behavioral, and neuropathological tests were also conducted. RESULTS Nerve conduction velocity and IL-10 concentration were increased in the antigen B group. The results of this study showed that antigen B reduced the inflammatory component of the EAE MS animal model by modulating the immune system compared to teriflunomide, which eventually led to a reduction in symptoms at the behavioral and electrophysiological level. CONCLUSIONS It seems that antigen B plays a critical role in regulating immunity and it can be used as a possible therapeutic agent to modulate the immune system in MS patients. It might be rational to consider hydatid cyst fluid antigen as a modifier in MS.
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Affiliation(s)
- Aliakbar Mariki
- Student Research Committee, Jiroft University of Medical Sciences, Jiroft, Iran
| | - Zahra Barzin
- Department of Parasitology and Mycology, School of Medicine, Jiroft University of Medical Science, Jiroft, Kerman, Iran
| | - Majid Fasihi Harandi
- Research Center for Hydatid Disease in Iran, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
| | | | - Mahboubeh Davoodi
- Student Research Committee, Yasuj University of Medical sciences, Yasuj, Iran
| | - Seyed Mohammad Mousavi
- Research Center for Hydatid Disease in Iran, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
| | - Soheila Rezakhani
- Neuroscience Research Center, Neuropharmacology Institute, Kerman University of Medical Sciences, Kerman, Iran
| | - Masoud Nazeri
- Neuroscience Research Center, Neuropharmacology Institute, Kerman University of Medical Sciences, Kerman, Iran.,Department of Anesthesiology, Friedrich-Alexander-University Erlangen-Nuremberg, University Hospital Erlangen, Krankenhausstraße, Germany
| | - Mohammad Shabani
- Neuroscience Research Center, Neuropharmacology Institute, Kerman University of Medical Sciences, Kerman, Iran
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Function of lipid binding proteins of parasitic helminths: still a long road. Parasitol Res 2022; 121:1117-1129. [PMID: 35169885 DOI: 10.1007/s00436-022-07463-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Accepted: 02/07/2022] [Indexed: 10/19/2022]
Abstract
Infections with parasitic helminths cause severe debilitating and sometimes lethal diseases in humans and domestic animals on a global scale. Unable to synthesize de novo their own fatty acids and sterols, helminth parasites (nematodes, trematodes, cestodes) rely on their hosts for their supply. These organisms produce and secrete a wide range of lipid binding proteins that are, in most cases, structurally different from the ones found in their hosts, placing them as possible novel therapeutic targets. In this sense, a lot of effort has been made towards the structure determination of these proteins, but their precise function is still unknown. In this review, we aim to present the current knowledge on the functions of LBPs present in parasitic helminths as well as novel members of this highly heterogeneous group of proteins.
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Biosa G, Bonelli P, Pisanu S, Ghisaura S, Santucciu C, Peruzzu A, Garippa G, Uzzau S, Masala G, Pagnozzi D. Proteomic characterization of Echinococcus granulosus sensu stricto, Taenia hydatigena and Taenia multiceps metacestode cyst fluids. Acta Trop 2022; 226:106253. [PMID: 34822852 DOI: 10.1016/j.actatropica.2021.106253] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 10/25/2021] [Accepted: 11/17/2021] [Indexed: 12/28/2022]
Abstract
Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.
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Affiliation(s)
- Grazia Biosa
- Porto Conte Ricerche Srl, Science and Technology Park of Sardinia, Tramariglio, Alghero, Sassari, Italy
| | - Piero Bonelli
- OIE Reference Laboratory for Echinococcosis, National Reference Center for Echinococcosis (CeNRE), IZS della Sardegna, Sassari, Italy
| | - Salvatore Pisanu
- Porto Conte Ricerche Srl, Science and Technology Park of Sardinia, Tramariglio, Alghero, Sassari, Italy.
| | - Stefania Ghisaura
- Porto Conte Ricerche Srl, Science and Technology Park of Sardinia, Tramariglio, Alghero, Sassari, Italy
| | - Cinzia Santucciu
- OIE Reference Laboratory for Echinococcosis, National Reference Center for Echinococcosis (CeNRE), IZS della Sardegna, Sassari, Italy
| | - Angela Peruzzu
- OIE Reference Laboratory for Echinococcosis, National Reference Center for Echinococcosis (CeNRE), IZS della Sardegna, Sassari, Italy
| | - Giovanni Garippa
- Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy
| | - Sergio Uzzau
- Porto Conte Ricerche Srl, Science and Technology Park of Sardinia, Tramariglio, Alghero, Sassari, Italy
| | - Giovanna Masala
- OIE Reference Laboratory for Echinococcosis, National Reference Center for Echinococcosis (CeNRE), IZS della Sardegna, Sassari, Italy
| | - Daniela Pagnozzi
- Porto Conte Ricerche Srl, Science and Technology Park of Sardinia, Tramariglio, Alghero, Sassari, Italy.
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Zhang H, Tian M, Qi W, Wu J, Zheng H, Guo G, Zhang L, Ranasinghe SL, McManus DP, Li J, Zhang W. Bioinformatic comparison of Kunitz protease inhibitors in Echinococcus granulosus sensu stricto and E. multilocularis and the genes expressed in different developmental stages of E. granulosus s.s. BMC Genomics 2021; 22:907. [PMID: 34922456 PMCID: PMC8684439 DOI: 10.1186/s12864-021-08219-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2021] [Accepted: 11/11/2021] [Indexed: 11/23/2022] Open
Abstract
Background
Cystic and alveolar echinococcosis caused by the tapeworms Echinococcus granulosus sensu stricto (s.s.) and E. multilocularis, respectively, are important zoonotic diseases. Protease inhibitors are crucial for the survival of both Echinococcus spp. Kunitz-type inhibitors play a regulatory role in the control of protease activity. In this study,we identified Kunitz-type domain protease inhibitors(KDPIs) present in the genomes of these two tapeworms and analyzed the gene sequences using computational, structural bioinformatics and phylogenetic approaches to evaluate the evolutionary relationships of these genes. Hi-seq transcriptome analysis showed that E. granulosuss.s. KDPIs were differentially expressed in the different developmental stages. We validated some of the genes expressed in adult worm, protoscolex and cyst germinal membrane of E. granulosuss.s. and E. multilocularis by quantitative PCR. Results A total of 19 genes from E. multilocularis and 23 genes from E. granulosuss.s. were predicted to be KDPIs with the most containing a single Kunitz-domain. A maximum likelihood method phylogenetic tree indicated that the E. granulosuss.s. and E. multilocularis Kunitz domain peptides were divided into three branches containing 9 clusters. The ratio of positively charged residues and neutral residues are different between E. multilocularis and E. granulosuss.s. KDPIs. We also found that E. multilocularis had higher percentage of sequences containing signal peptides (17/19, 89.47%) than that of E. granulosuss.s. (14/23, 60.87%). Transcript analysis showed all the E. granulosuss.s. KDPI genes were expressed differentially in four developmental stages of the worm. Transcription analysis showed that 9 KDPIs (including EG_07244,EGR_08716 and EGR_10096) were highly upregulated in adult worm, and 2 KDPIs (EG_09268 and EG_09490) were highly expressed in the cyst germinal membrane. Quantitative gene expression analysis(qPCR) of four genes confirmed the expression of these genes. EGR_08716 and its homologous gene (EmuJ_001137000) were highly and specifically expressed in adult worms of the two worms. Conclusions A total 19 and 23 KDPIs were identified in the genomes of E. multilocularis and E. granulosus s.s. , respectively. The differential expression of these KDPIs in different stages may indicate their different roles in the different hosts. The difference in characterization of KDPIs may be associated with the different pathology of metacestode stage of these two parasites. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-08219-4.
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Sánchez-Ovejero C, Akdur E, Manzano-Román R, Hernández-González A, González-Sánchez M, Becerro-Recio D, González-Miguel J, Akhan O, Cretu CM, Vutova K, Tamarozzi F, Mariconti M, Brunetti E, Vola A, Fabiani M, Casulli A, Siles-Lucas M. Evaluation of the sensitivity and specificity of GST-tagged recombinant antigens 2B2t, Ag5t and DIPOL in ELISA for the diagnosis and follow up of patients with cystic echinococcosis. PLoS Negl Trop Dis 2020; 14:e0008892. [PMID: 33253168 PMCID: PMC7728171 DOI: 10.1371/journal.pntd.0008892] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Revised: 12/10/2020] [Accepted: 10/14/2020] [Indexed: 12/23/2022] Open
Abstract
Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosis and monitoring of CE rely primarily on imaging while serology is used as a confirmatory test. However, imaging is not always conclusive and currently available serological assays have suboptimal sensitivity and specificity, lack standardization, and are not useful for patients´ follow-up. Seroassays for CE are usually based on hydatid fluid (HF), a complex, variable antigenic mixture, and cross-reactivity exists especially with alveolar echinococcosis. Recombinant proteins based on immunogenic antigens most abundant in HF, such as AgB1, AgB2 and Ag5, have been used to overcome these limitations. None of them so far showed potential to replace HF; however, their performance have been largely tested on a limited number of samples, and comparison of different antigens using the same cohort has been rarely performed. The combination of several immunogenic epitopes in a single recombinant protein could enhance test sensitivity. For the diagnosis and follow-up of patients with CE, we compared the performance of the crude HF, previously described recombinant 2B2t antigen, and GST-tagged version of 2B2t, and novel designed recombinants (GST-Ag5t and the GST-DIPOL chimera containing AgB1, AgBB2 and Ag5 epitopes) by IgG-ELISA format. Samples belong to a retrospective cohort of 253 well-characterized patients with CE, previously described for the evaluation of the 2B2t antigen, 92 patients with alveolar echinococcosis, and 82 healthy donors. The reference standard for CE diagnosis was the presence of a CE lesion as diagnosed by ultrasonography. The highest sensitivity was obtained with HF [86.7%, 95% confidence interval (CI): 81.2–91.0], followed by GST-2B2t (70.0%, 95% CI: 63.1–76.2), 2B2t (65.5%, 95% CI: 58.5–72.0), GST-Ag5t (64.5%, 95% CI: 57.5–71.1) and GST-DIPOL (63.1%, 95% CI: 56.0–69.7). The GST-2B2t had the best specificity (95.8%, 95% CI: 88.3–99.1) and the lowest cross-reactivity (38.7%, 95% CI: 27.6–50.6). Good response to treatment also correlated to negative test results in the GST-2B2t ELISA. While none of the tested recombinant antigen appears suitable to replace HF for the diagnosis of CE, GST-2B2t should be further explored as a confirmation test, based on its high specificity and low cross-reactivity, and for the follow-up after treatment in those patients with positive serology for this antigen. Cystic echinococcosis (CE) is a neglected parasitic zoonosis. Its diagnosis and follow-up require evaluation with imaging. Currently available serological tests are applied to confirm the diagnosis in doubtful cases, although having limitations in diagnostic accuracy, and they are not useful for patients’ follow-up. Seroassays for CE are usually based on hydatid fluid (HF) obtained from infected animals, with consequent problems of heterogeneity and low specificity. The use of semi-purified HF derivatives or recombinant antigens has been attempted to improve these aspects, but with an unacceptable loss in sensitivity. Most newly developed antigens have been tested on a limited number of samples, not always well characterized, and have been rarely compared using the same samples cohort. Here, we tested and compared three recombinant antigens (2B2t, GST-2B2t and GST-Ag5t), and a recombinant chimeric antigen (DIPOL) based on three highly immunogenic components of HF (B1, B2 and Ag5), in an attempt to increase the sensitivity of recombinant antigen-based seroassays for the diagnosis and follow-up of patients with CE. We found that GST-2B2t had higher sensitivity than the other antigenic preparations, but still not as high as HF, and that GST-2B2t and GST-DIPOL had statistically higher specificity than any of the other tested antigens. GST-2B2t also showed potential for the follow-up of patients with CE after drug treatment.
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Affiliation(s)
| | - Eylem Akdur
- Cukurova Univeristy, Department of Parasitology, Sarıçam/Adana, Turkey
| | - Raúl Manzano-Román
- Proteomic Unit, Center for Cancer Research, University of Salamanca, Campus Miguel de Unamuno, Salamanca
| | - Ana Hernández-González
- Instituto de Salud Carlos III, Centro Nacional de Microbiología, Majadahonda, Madrid, Spain
| | - María González-Sánchez
- Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas, Salamanca, Spain
| | - David Becerro-Recio
- Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas, Salamanca, Spain
| | - Javier González-Miguel
- Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas, Salamanca, Spain
| | - Okan Akhan
- Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Carmen M. Cretu
- University of Medicine and Pharmacy, Colentina Clinical Hospital—Parasitology, Bucharest, Romania
| | - Kamenna Vutova
- Specialised Hospital of Infectious and Parasitic Diseases "Prof. Ivan Kirov", Department of Infectious, Parasitic and Tropical Diseases, Medical University, Sofia, Bulgaria
| | - Francesca Tamarozzi
- WHO Collaborating Centre for the epidemiology, detection and control of cystic and alveolar echinococcosis, Istituto Superiore di Sanità, Rome, Italy
| | - Mara Mariconti
- Department of Clinical Surgical Diagnostic and Paediatric Sciences, University of Pavia, Via Taramelli 5, Pavia, Italy
| | - Enrico Brunetti
- Department of Clinical Surgical Diagnostic and Paediatric Sciences, University of Pavia, and Division of Infectious and Tropical Diseases, San Matteo Hospital Foundation, Via Taramelli 5, Pavia, Italy
| | - Ambra Vola
- San Matteo Hospital Foundation, Via Taramelli 5, Pavia, Italy
| | - Massimo Fabiani
- Infectious Diseases Department, Istituto Superiore di Sanità, Rome, Italy
| | - Adriano Casulli
- WHO Collaborating Centre for the epidemiology, detection and control of cystic and alveolar echinococcosis, Istituto Superiore di Sanità, Rome, Italy
- European Reference Laboratory for Parasites (EURLP), Istituto Superiore di Sanità, Rome, Italy
| | - Mar Siles-Lucas
- Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Cordel de Merinas, Salamanca, Spain
- * E-mail:
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Identification of antigen B (AgB) Gene polymorphism in cattle and sheep isolates of Echinococcus granulosus and investigation of effects on serological diagnosis. Acta Trop 2019; 199:105099. [PMID: 31356785 DOI: 10.1016/j.actatropica.2019.105099] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2019] [Revised: 07/16/2019] [Accepted: 07/17/2019] [Indexed: 11/24/2022]
Abstract
Cystic Echinococcosis (CE) is a worldwide common helminth disease caused by the larval form of Echinococcus granulosus. The aim of this study is to determine the genetic differences between distinct isolates of E. granulosus obtained from cattle and sheep and determine the polymorphism of the AgB1 gene by DNA sequence analysis, as well as investigating its relationship with serological response using ELISA and Western Blot tests. For this aim, germinal membranes of hydatid cysts of 30 cattle and 30 sheep from the provinces of Elazig and Erzincan in Turkey and serum samples of these animals were collected. Following isolation of the total genomic DNA, the 12S rRNA gene of all isolates was amplified by PCR for genetic characterization, and the mt-CO1 gene region was examined by DNA sequence analysis. The gDNAs were then amplified by PCR using AgB1-specific primers, and genetic variation was investigated by DNA sequence analysis. At the final stage, all serum samples were analyzed by ELISA and Western Blot tests using a partially purified hydatid cyst fluid antigen. As a result, 114 (95%) of the 120 isolates were determined to be E. granulosus sensu stricto by using 12S rRNA-PCR. Subsequently, the DNA sequence analysis of the remaining 6 samples of the mt-CO1 gene revealed that all samples were E. granulosus sensu stricto. According to the DNA sequence analysis that followed, nucleotide changes in the AgB1 gene were observed in 13 (10.8%) of 120 samples. With this study, 9 (69.2%) out of 13 hydatid cysts in which polymorphism was detected by DNA sequence analysis in their AgB1 gene were found to be positive with ELISA, and 6 (46.1%) were found positive by WB. While 80 (74.7%) of 107 non-polymorphic samples in the AgB1 gene were found to be positive with ELISA, and 75 (70.9%) were positive with WB. As a result, variation in different ratios was determined in the AgB1 gene of E. granulosus sensu stricto, and it was determined that this had a partial effect on serological response.
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11
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Liu Y, Yang Y, Xu J, Dong X, Gu X, Xie Y, Lai W, Jing B, Peng X, Yang G. Expression and serodiagnostic potential of antigen B and thioredoxin peroxidase from Taenia multiceps. Vet Parasitol 2019; 272:58-63. [PMID: 31395206 DOI: 10.1016/j.vetpar.2019.07.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2019] [Revised: 07/08/2019] [Accepted: 07/11/2019] [Indexed: 10/26/2022]
Abstract
Coenurosis is a serious parasitic disease of herbivorous animals caused by the metacestode of Taenia multiceps (Coenurus cerebralis). Accordingly, a significant amount of research is currently dedicated to the development of appropriate antigens for use in rapid and accurate coenurosis diagnosis kits. In the present study, antigen B (AgB) and thioredoxin peroxidase (TPx) from T. multiceps were cloned and expressed using a prokaryotic system, molecular characterization of Tm-AgB was determined by bioinformatical analyses. The serological diagnostic potentials of rTm-AgB and rTm-TPx were evaluated by indirect ELISA and compared with those of previously reported rTm-AnxB2, rTm-HSP70, and rTm-GST. The results showed that Tm-AgB is a specific lipoprotein of cestodes with good thermal stability. The ELISA assay showed that rTm-AgB exhibited a sensitivity of 95.8% and a specificity of 87.5%, indicating its strong potential for serological diagnosis of T. multiceps.
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Affiliation(s)
- Yuchen Liu
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Yingdong Yang
- Panzhihua Academy of Agricultural and Forestry Sciences, Panzhihua 617000, China.
| | - Jing Xu
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Xiaowei Dong
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Xiaobin Gu
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Yue Xie
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Weimin Lai
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Bo Jing
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Xuerong Peng
- Department of Chemistry, College of Life and Basic Science, Sichuan Agricultural University, Wenjiang 611130, China.
| | - Guangyou Yang
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China.
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12
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Han X, Kim JG, Wang H, Cai H, Ma X, Duong DH, Ahn CS, Kang I, Kong Y. Survey of echinococcoses in southeastern Qinghai Province, China, and serodiagnostic insights of recombinant Echinococcus granulosus antigen B isoforms. Parasit Vectors 2019; 12:323. [PMID: 31242932 PMCID: PMC6593596 DOI: 10.1186/s13071-019-3569-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2018] [Accepted: 06/17/2019] [Indexed: 12/13/2022] Open
Abstract
Background Echinococcoses, caused by metacestodes of Echinococcus granulosus (cystic echinococcosis; CE) and E. multilocularis (alveolar echinococcosis; AE), represent major emerging parasitic diseases. These enzootic helminthiases invoke significant public health concerns and social burdens in endemic areas. The diseases are prevalent in the Qinghai-Tibetan Plateau, China, while community-based epidemiological studies have been scarcely reported. We surveyed echinococcosis patients in the southeastern Qinghai Province, China, to better understand the concurrent epidemiological situation in this area. Methods During July and August of 2013 and 2014, we screened echinococcosis patients at Yushu and Golog Prefectures, Qinghai Province, China, in a diagnostic campaign. A total of 2856 people (male:female ratio, 1:1.12; mean age, 34.6 years; age range, 6–88 years) were ultrasonographically examined for the presence of hepatic echinococcal cysts. We also collected serum samples from patients and analyzed antibody reactivity against recombinant forms of diverse E. granulosus antigen Bs (rEgAgB1-5) by enzyme-linked immunosorbent assay. Results We detected 134 patients whose imaging scans were compatible with CE (115 cases) and AE (20 patients). One patient might have been infected with both CE and AE. The overall incidence was 4.7% (CE, 4.0%; AE, 0.7%). A large proportion (67.5%) of CE patients was diagnosed at active and transitional CE1-CE3 stages in their late 30s. The AE cases were generally detected at advanced stage in patients at early 20s (60%). Analysis of the receiver operating characteristic curve and Youden’s index indicated that rEgAgB2 was the most promising biomarker, followed by rEgAgB3 and rEgAgB1. Overall, sensitivity and specificity of rEgAgB1-3 were 84.5–92.7% and 91.9–94.6%, respectively. rEgAgB4 and 5 showed low sensitivity with high cross-reactivity. Conclusions Our results strongly suggest that disability-adjusted life years related to echinococcoses in Qinghai-Tibetan areas might be more serious than previously considered. Control and prevention strategy against CE and AE are highly required in these areas. In addition to ultrasonography, serological tests might provide supportive data. However, serological data should be carefully interpreted for differential diagnosis, especially in areas where both CE and AE are co-endemic. Electronic supplementary material The online version of this article (10.1186/s13071-019-3569-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Xiumin Han
- Qinghai Provincial People's Hospital, Xining, 810007, China.,Qinghai Province Institute for Endemic Diseases Prevention and Control, Qinghai Centers for Disease Prevention and Control, Xining, 811602, China
| | - Jeong-Geun Kim
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, 16419, Korea
| | - Hu Wang
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Qinghai Centers for Disease Prevention and Control, Xining, 811602, China.,Endemic Disease Administration Office, Qinghai Province Health and Family Planning Commission, Xining, 811602, China
| | - Huixia Cai
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Qinghai Centers for Disease Prevention and Control, Xining, 811602, China.,Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, 16419, Korea
| | - Xiao Ma
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Qinghai Centers for Disease Prevention and Control, Xining, 811602, China
| | - Duc Hieu Duong
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, 16419, Korea
| | - Chun-Seob Ahn
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, 16419, Korea
| | - Insug Kang
- Department of Molecular Biology and Biochemistry, Kyung Hee University College of Medicine, Seoul, 02447, Korea
| | - Yoon Kong
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, 16419, Korea.
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Ebrahimipour M, Afgar A, Barati M, Mohammadi MA, Harandi MF. Evaluation of the antigenic epitopes of EgAgB/1 and EgAgB/4 subunit antigens in G1 and G6 genotypes of Echinococcus granulosus using bioinformatics. GENE REPORTS 2019. [DOI: 10.1016/j.genrep.2019.01.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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14
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da Silva ED, Cancela M, Monteiro KM, Ferreira HB, Zaha A. Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways. PLoS Negl Trop Dis 2018; 12:e0006473. [PMID: 29727452 PMCID: PMC5955594 DOI: 10.1371/journal.pntd.0006473] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2017] [Revised: 05/16/2018] [Accepted: 04/23/2018] [Indexed: 01/18/2023] Open
Abstract
Background Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid) of Echinococcus granulosus (Cestoda, Taeniidae). The hydatid develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. The lipoprotein Antigen B (AgB) is the major component of E. granulosus metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known. Methodology/Principal findings In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of caveolae/raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. Finally, AgB colocalized with an endolysosomal tracker, providing evidence for a possible AgB destination after endocytosis. Conclusions/Significance The results indicate that caveolae/raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. A possible fate for AgB after endocytosis seems to be the endolysosomal system. Cellular internalization and further access to subcellular compartments could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival. Antigen B (AgB) is an oligomeric lipoprotein highly abundant in Echinococcus granulosus hydatid fluid. AgB has already been characterized as an immunomodulatory protein, capable of inducing a permissive immune response to parasite development. Also, an important role in lipid acquisition is attributed to AgB, because it has been found associated to different classes of host lipids. However, the mechanisms of interaction employed by AgB to perform its functions remain undetermined. In this study, we demonstrate that mammalian cells are able to internalize E. granulosus AgB in culture and found that specific mechanisms of endocytosis are involved. Our results extend the understanding of AgB biological role indicating cellular internalization as a mechanism of interaction, which in turn, may represent a target to intervention.
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Affiliation(s)
- Edileuza Danieli da Silva
- Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Martin Cancela
- Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Karina Mariante Monteiro
- Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Henrique Bunselmeyer Ferreira
- Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Arnaldo Zaha
- Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- * E-mail:
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Fatemi Esfedan A, Sarkari B, Mikaeili F. Genetic Variability of Antigen B8/1 among Echinococcus granulosus Isolates from Human, Cattle, and Sheep in Fars Province, Southern Iran. Rep Biochem Mol Biol 2018; 6:164-160. [PMID: 29765999 PMCID: PMC5941130] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2017] [Accepted: 08/03/2017] [Indexed: 06/08/2023]
Abstract
BACKGROUND Cystic echinococcosis (CE), known as hydatid cyst, is a zoonotic parasitic infection caused by the larval stage of Echinococcus granulosus (E. granulosus). Antigen B, the major component of hydatid cyst fluid, is encoded by members of a multigene family. The present study aimed to evaluate the genetic diversity of the gene encoding antigen B8/1 (EgAgB8/1) among the main intermediate hosts of E. granulosus. METHODS Twenty-eight hydatid cyst isolates (10 sheep, 9 human, and 9 cattle) were collected in Fars province, Iran. DNA was extracted from each cyst and PCR, followed by DNA sequencing was used to identify potential EgAgB8/1 sequence variation and polymorphism. A phylogenetic tree was constructed using MEGA 7.0 software and the maximum likelihood method. RESULTS Using EgAgB8/1 primers, an approximately 315 bp band was amplified from all the isolates. The PCR products were sequenced, and the sequences were deposited in GenBank (accession numbers, KY709266-KY709293). The polymorphism variation among the isolates was 0.0, while intra-species variation within the isolates and related sequences in GenBank was 0.5-1%. Analysis of the phylogenetic tree revealed that the isolates from humans, sheep, and cattle all cluster in one group and are homologous to the EgAgB8/1 M1 allele. CONCLUSION Findings of this study revealed close similarity between the EgAgB8/1 of human, sheep, and cattle E. granulosus isolates. However, differences were found between the EgAgB8/1 sequences in our study and those reported from other CE endemic areas. Whether such similarities and differences exist in other subunits AgB subunits require further study.
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Affiliation(s)
- Asieh Fatemi Esfedan
- Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Bahador Sarkari
- Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
- Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Fataneh Mikaeili
- Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
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Grubor NM, Jovanova-Nesic KD, Shoenfeld Y. Liver cystic echinococcosis and human host immune and autoimmune follow-up: A review. World J Hepatol 2017; 9:1176-1189. [PMID: 29109850 PMCID: PMC5666304 DOI: 10.4254/wjh.v9.i30.1176] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2017] [Revised: 08/28/2017] [Accepted: 09/14/2017] [Indexed: 02/06/2023] Open
Abstract
Cystic echinococcosis (CE) is an infectious disease caused by the larvae of parasite Echinococcus granulosus (E. granulosus). To successfully establish an infection, parasite release some substances and molecules that can modulate host immune functions, stimulating a strong anti-inflammatory reaction to carry favor to host and to reserve self-survival in the host. The literature was reviewed using MEDLINE, and an open access search for immunology of hydatidosis was performed. Accumulating data from animal experiments and human studies provided us with exciting insights into the mechanisms involved that affect all parts of immunity. In this review we used the existing scientific data and discuss how these findings assisted with a better understanding of the immunology of E. granulosus infection in man. The aim of this study is to point the several facts that challenge immune and autoimmune responses to protect E. granulosus from elimination and to minimize host severe pathology. Understanding the immune mechanisms of E. granulosus infection in an intermediate human host will provide, we believe, a more useful treatment with immunomodulating molecules and possibly better protection from parasitic infections. Besides that, the diagnosis of CE has improved due to the application of a new molecular tool for parasite identification by using of new recombinant antigens and immunogenic peptides. More studies for the better understanding of the mechanisms of parasite immune evasion is necessary. It will enable a novel approach in protection, detection and improving of the host inflammatory responses. In contrast, according to the "hygiene hypothesis", clinical applications that decrease the incidence of infection in developed countries and recently in developing countries are at the origin of the increasing incidence of both allergic and autoimmune diseases. Thus, an understanding of the immune mechanisms of E. granulosus infection is extremely important.
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Affiliation(s)
- Nikica M Grubor
- Department of Hepatobiliary and Pancreatic Surgery, First Surgical University Hospital, Clinical Center of Serbia, School of Medicine University of Belgrade, 11000 Belgrade, Serbia
| | - Katica D Jovanova-Nesic
- Immunology Research Center, Institute of Virology, Vaccine and Sera-Torlak, 11221 Belgrade, Serbia
- European Center for Peace and Development, University for Peace in the United Nation established in Belgrade, 11000 Belgrade, Serbia.
| | - Yehuda Shoenfeld
- Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel Aviv University, 5265601 Tel-Hashomer, Tel Aviv, Israel
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17
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Yang M, Li J, Wu J, Wang H, Guo B, Wu C, Shou X, Yang N, Zhang Z, McManus DP, Zhang F, Zhang W. Cloning and characterization of an Echinococcus granulosus ecdysteroid hormone nuclear receptor HR3-like gene. ACTA ACUST UNITED AC 2017; 24:36. [PMID: 28971798 PMCID: PMC5625357 DOI: 10.1051/parasite/2017037] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2017] [Accepted: 09/04/2017] [Indexed: 12/13/2022]
Abstract
Cystic echinococcosis is an important parasitic zoonosis caused by the dog tapeworm Echinococcus granulosus. Little is known about adult worm development at the molecular level. Transcription analysis showed that the E. granulosus hormone receptor 3-like (EgHR3) gene was expressed in protoscoleces and adult worms, indicating its role in early adult development. In this study, we cloned and characterized EgHR3 showing that its cDNA contains an open reading frame (ORF) of 1890 bp encoding a 629 amino acid protein, which has a DNA-binding domain (DBD) and a ligand-binding domain (LBD). Immunolocalization revealed the protein was localized in the parenchyma of protoscoleces and adult worms. Real-time PCR analysis showed that EgHR3 was expressed significantly more in adults than in other stages of development (p<0.01) and that its expression was especially high in the early stage of adult worm development induced by bile acids. EgHR3 siRNA silenced 69–78% of the level of transcription in protoscoleces, which resulted in killing 43.6–60.9% of protoscoleces after 10 days of cultivation in vitro. EgHR3 may play an essential role in early adult worm development and in maintaining adult biological processes and may represent a novel drug or vaccine target against echinococcosis.
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Affiliation(s)
- Mei Yang
- Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 14 Shengli Road, Urumqi 830046, PR China - Basic Medical College of Xinjiang Medical University, Urumqi 830011, PR China
| | - Jun Li
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
| | - Jun Wu
- Public Health College of Xinjiang Medical University, Urumqi 830011, PR China
| | - Hui Wang
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
| | - Baoping Guo
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
| | - Chuanchuan Wu
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
| | - Xi Shou
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
| | - Ning Yang
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
| | - Zhuangzhi Zhang
- Molecular Parasitology Laboratory, QIMR Berghofer, Herston, QLD, 4006, Australia
| | - Donald P McManus
- Veterinary Research Institute, Xinjiang Academy of Animal Sciences, Urumqi 830000, PR China
| | - Fuchun Zhang
- Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 14 Shengli Road, Urumqi 830046, PR China
| | - Wenbao Zhang
- State Key Laboratory of Pathogenesis, Prevention and Treatment of Central Asian High Incidence Diseases, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, PR China
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Ahn CS, Kim JG, Han X, Kang I, Kong Y. Comparison of Echinococcus multilocularis and Echinococcus granulosus hydatid fluid proteome provides molecular strategies for specialized host-parasite interactions. Oncotarget 2017; 8:97009-97024. [PMID: 29228589 PMCID: PMC5722541 DOI: 10.18632/oncotarget.20761] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2017] [Accepted: 08/09/2017] [Indexed: 12/16/2022] Open
Abstract
Alveolar and cystic echinococcoses, caused by the metacestodes of Echinococcus multilocularis and E. granulosus, are prevalent in several regions and invoke deleterious zoonotic helminthiases. Hydatid fluid (HF), which contains proteinaceous and non-proteinaceous secretions of the parasite- and host-derived components, critically affects the host-parasite interplay and disease progression. We conducted HF proteome profiling of fully mature E. multilocularis vesicle (nine months postinfection) and E. granulosus cyst (stage 2). We identified 120 and 153 proteins, respectively, in each fluid. Fifty-six and 84 proteins represented distinct species; 44 and 66 were parasites, and 12 and 18 were host-derived proteins. The five major parasite protein populations, which included antigen B isoforms, metabolic enzymes, proteases and inhibitors, extracellular matrix molecules (ECMs), and developmental proteins, were abundantly distributed in both fluids and also exclusively in one sample or the other. Carbohydrate-metabolizing enzymes were enriched in E. granulosus HF. In the E. multilocularis HF, proteins that constitute ECMs, which might facilitate adhesion and cytogenesis, were highly expressed. Those molecules had physical and functional relationships along with their biochemical properties through protein-protein interaction networks. Twelve host-derived proteins were largely segregated to serum components. The major proteins commonly and uniquely detected in these HFs and their symbiotic interactome relationships might reflect their biological roles in similar but distinct modes of maturation, invasion, and the longevity of the parasites in the hosts.
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Affiliation(s)
- Chun-Seob Ahn
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, Korea
| | - Jeong-Geun Kim
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, Korea
| | - Xiumin Han
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Xining, China.,Clinical Research Institute for Hydatid Disease, Qinghai Provincial People's Hospital, Xining, China
| | - Insug Kang
- Department of Molecular Biology and Biochemistry, Kyung Hee University School of Medicine, Seoul, Korea
| | - Yoon Kong
- Department of Molecular Parasitology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Suwon, Korea
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Monteiro KM, Lorenzatto KR, de Lima JC, Dos Santos GB, Förster S, Paludo GP, Carvalho PC, Brehm K, Ferreira HB. Comparative proteomics of hydatid fluids from two Echinococcus multilocularis isolates. J Proteomics 2017; 162:40-51. [PMID: 28442449 DOI: 10.1016/j.jprot.2017.04.009] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2016] [Revised: 03/21/2017] [Accepted: 04/10/2017] [Indexed: 02/06/2023]
Abstract
The hydatid fluid (HF) that fills Echinococcus multilocularis metacestode vesicles is a complex mixture of proteins from both parasite and host origin. Here, a LC-MS/MS approach was used to compare the HF composition of E. multilocularis H95 and G8065 isolates (EmH95 and EmG8065, respectively), which present differences in terms of growth and fertility. Overall, 446 unique proteins were identified, 392 of which (88%) were from parasite origin and 54 from culture medium. At least 256 of parasite proteins were sample exclusive, and 82 of the 136 shared proteins presented differential abundance between E. multilocularis isolates. The parasite's protein repertoires in EmH95 and EmG8065 HF samples presented qualitative and quantitative differences involving antigens, signaling proteins, proteolytic enzymes, protease inhibitors and chaperones, highlighting intraspecific singularities that could be correlated to biological features of each isolate. The repertoire of medium proteins found in the HF was also differential between isolates, and the relevance of the HF exogenous protein content for the parasite's biology is discussed. The repertoires of identified proteins also provided potential molecular markers for important biological features, such as parasite growth rate and fertility, as well potential protein targets for the development of novel diagnostic and treatment strategies for alveolar echinococcosis. BIOLOGICAL SIGNIFICANCE E. multilocularis metacestode infection of mammal hosts involve complex interactions mediated by excretory/secretory (ES) products. The hydatid fluid (HF) that fills the E. multilocularis metacestode vesicles contains complex repertoires of parasite ES products and host proteins that mediate important molecular interactions determinant for parasite survival and development, and, consequently, to the infection outcome. HF has been also extensively reported as the main source of proteins for the immunodiagnosis of echinococcosis. The performed proteomic analysis provided a comprehensive profiling of the HF protein composition of two E. multilocularis isolates. This allowed us to identify proteins of both parasite and exogenous (medium) origin, many of which present significant differential abundances between parasite isolates and may correlate to their differential biological features, including fertility and growth rate.
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Affiliation(s)
- Karina M Monteiro
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Departamento de Biologia Molecular e Celular, Instituto de Biociências, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil
| | - Karina R Lorenzatto
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil
| | - Jeferson C de Lima
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil
| | - Guilherme B Dos Santos
- Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil
| | - Sabine Förster
- University of Würzburg, Institute of Hygiene and Microbiology, Würzburg, Germany
| | - Gabriela P Paludo
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil
| | - Paulo C Carvalho
- Laboratório de Proteômica e Engenharia de Proteínas, Instituto Carlos Chagas, FIOCRUZ, Curitiba, PR, Brazil
| | - Klaus Brehm
- University of Würzburg, Institute of Hygiene and Microbiology, Würzburg, Germany
| | - Henrique B Ferreira
- Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Departamento de Biologia Molecular e Celular, Instituto de Biociências, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil.
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20
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Al-Kitani FA, Mansoor MK, Hussain MH, Al Rawahi AH, Saqib M, Al Maawali MG. Sero-epidemiology of cystic echinococcosis (Echinococcus granulosus) in the livestock of Oman. VETERINARY PARASITOLOGY- REGIONAL STUDIES AND REPORTS 2017; 8:21-27. [PMID: 31014632 DOI: 10.1016/j.vprsr.2017.01.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Revised: 12/03/2016] [Accepted: 01/06/2017] [Indexed: 10/20/2022]
Abstract
A cross-sectional serological survey of cystic echinocossis was carried out on 2802 randomly collected sera of camels (n=706), cattle (n=687), goats (n=701) and sheep (n=708) from all governorates of Oman. The samples were analysed by in house indirect ELISA (iELISA) using Echinococcus granulosus antigen B (EgAgB) of naturally infected camels. The overall percentage of antibodies against EgAgB was found to be 14.6%. The highest percentage of positive was observed in sera from camels (22.4%) followed by cattle (12.9%), sheep (12.2%) and goats (10.9%). The highest percentage of seropositivity was observed in females (15.4%) as compared to male animals (10.6%). The imported livestock were found more seropositive (15.2%) as compared to local (14.7%) and crossbred livestock (14.1%). The highest exposure was observed in animals with the age group of above 5years (18.3%) followed by the age group of up to 2years (15.1%,) and those between 2 and 5years (12.4%). The univariate analysis has indicated that camels (OR:2.33, CI 1.74, 3.14), cattle (OR:1.21, CI 0.87, 1.67), sheep (OR:1.12, CI 0.81, 1.55) were more likely to test positive than goats. Furthermore, females (OR: 1.53, CI 1.11, 2.11) were more likely to test seropositive. Sera from animals above 5years of age (OR:1.58, CI 1.25, 2.01) and between 2 and 5years old (OR:1.30, CI 0.98, 1.71) were found more likely to test seropositive than those up to 2years of age. The multivariable analysis at individual level indicated that camels (OR: 2.07, CI 1.66, 2.56, p<0.001) and female (OR: 1.43, CI 1.04, 1.98, p=0.030) were more likely to acquire CE. At herd level, the final multivariable model indicated that herds located in Dofar and Musandam (OR: 4.48, CI 2.69, 7.45, p<0.001), in areas receiving seasonal rains (OR: 2.54, CI 1.09, 5.90, p<0.001) and practicing transhumance (OR: 4.22, CI 1.84, 9.65, p<0.001) and sedentary (OR: 2.07, CI 1.38, 3.12, p=0.001) farming system were more likely to acquire CE in Oman. The study documents the serological evidence of CE in livestock of Oman and a carefully planned control program should be devised after further epidemiological and molecular investigations in the intermediate and final hosts.
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Affiliation(s)
| | | | - Muhammad Hammad Hussain
- Animal Health Research Centre, Ministry of Agriculture & Fisheries, Oman; Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan
| | | | - Muhammad Saqib
- Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan
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Laboratory Diagnosis of Echinococcus spp. in Human Patients and Infected Animals. ADVANCES IN PARASITOLOGY 2017; 96:159-257. [PMID: 28212789 DOI: 10.1016/bs.apar.2016.09.003] [Citation(s) in RCA: 121] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Among the species composing the genus Echinococcus, four species are of human clinical interest. The most prevalent species are Echinococcus granulosus and Echinococcus multilocularis, followed by Echinococcus vogeli and Echinococcus oligarthrus. The first two species cause cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively. Both diseases have a complex clinical management, in which laboratory diagnosis could be an adjunctive to the imaging techniques. To date, several approaches have been described for the laboratory diagnosis and followup of CE and AE, including antibody, antigen and cytokine detection. All of these approaches are far from being optimal as adjunctive diagnosis particularly for CE, since they do not reach enough sensitivity and/or specificity. A combination of several methods (e.g., antibody and antigen detection) or of several (recombinant) antigens could improve the performance of the adjunctive laboratory methods, although the complexity of echinococcosis and heterogeneity of clinical cases make necessary a deep understanding of the host-parasite relationships and the parasite phenotype at different developmental stages to reach the best diagnostic tool and to make it accepted in clinical practice. Standardization approaches and a deep understanding of the performance of each of the available antigens in the diagnosis of echinococcosis for the different clinical pictures are also needed. The detection of the parasite in definitive hosts is also reviewed in this chapter. Finally, the different methods for the detection of parasite DNA in different analytes and matrices are also reviewed.
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22
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Folle AM, Kitano ES, Lima A, Gil M, Cucher M, Mourglia-Ettlin G, Iwai LK, Rosenzvit M, Batthyány C, Ferreira AM. Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype. PLoS Negl Trop Dis 2017; 11:e0005250. [PMID: 28045899 PMCID: PMC5234841 DOI: 10.1371/journal.pntd.0005250] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Revised: 01/13/2017] [Accepted: 12/12/2016] [Indexed: 12/23/2022] Open
Abstract
The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed. Cystic echinococcosis (CE), a worldwide-spread zoonosis, affects livestock mammals and humans with significant economic and public health impact. It is caused by the infection with the larva of cestodes belonging to Echinococcus granulosus complex, a series of parasite species with preference for different hosts. Among them, Echinococcus canadensis larva uses mainly camels, goats and pigs as hosts. Species/genotypes belonging to E. canadensis are considered the second most common cause of human CE, but its contribution may be underestimated since causes asymptomatic or more benign infections than other E. granulosus complex species. The most relevant antigen for CE diagnosis is a lipoprotein called antigen B (AgB). AgB antigenicity is linked to its protein moiety that is encoded by several genes. One of these genes, AgB2, seems to be differentially expressed within E. granulosus complex. Using high sensitivity proteomic tools we analysed the composition of AgB obtained from E. canadensis larva, detecting the protein products of all AgB genes, except AgB2. Since AgB subunits have been widely used as antigens in immunoassays for human CE diagnosis, our results indicate that using AgB2 protein product in these assays may lead to false-negative results, particularly in geographical areas where E. canadensis species/genotypes circulate.
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Affiliation(s)
- Ana Maite Folle
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Eduardo S. Kitano
- Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan São Paulo, Brazil
| | - Analía Lima
- Unidad de Bioquímica y Proteómica Analíticas, Instituto Pasteur de Montevideo, Montevideo, Uruguay
| | - Magdalena Gil
- Unidad de Bioquímica y Proteómica Analíticas, Instituto Pasteur de Montevideo, Montevideo, Uruguay
| | - Marcela Cucher
- Instituto de Investigaciones en Microbiología y Parasitología Médica, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Gustavo Mourglia-Ettlin
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Leo K. Iwai
- Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan São Paulo, Brazil
| | - Mara Rosenzvit
- Instituto de Investigaciones en Microbiología y Parasitología Médica, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Carlos Batthyány
- Unidad de Bioquímica y Proteómica Analíticas, Instituto Pasteur de Montevideo, Montevideo, Uruguay
- Departamento de Bioquímica, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Ana María Ferreira
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
- * E-mail:
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23
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Díaz A, Casaravilla C, Barrios AA, Ferreira AM. Parasite molecules and host responses in cystic echinococcosis. Parasite Immunol 2016; 38:193-205. [PMID: 26425838 DOI: 10.1111/pim.12282] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Accepted: 09/22/2015] [Indexed: 01/03/2023]
Abstract
Cystic echinococcosis is the infection by the larvae of cestode parasites belonging to the Echinococcus granulosus sensu lato species complex. Local host responses are strikingly subdued in relation to the size and persistence of these larvae, which develop within mammalian organs as 'hydatid cysts' measuring up to tens of cm in diameter. In a context in which helminth-derived immune-suppressive, as well as Th2-inducing, molecules garner much interest, knowledge on the interactions between E. granulosus molecules and the immune system lags behind. Here, we discuss what is known and what are the open questions on E. granulosus molecules and structures interacting with the innate and adaptive immune systems, potentially or in demonstrated form. We attempt a global biological approach on molecules that have been given consideration primarily as protective (Eg95) or diagnostic antigens (antigen B, antigen 5). We integrate glycobiological information, which traverses the discussions on antigen 5, the mucin-based protective laminated layer and immunologically active preparations from protoscoleces. We also highlight some less well-known molecules that appear as promising candidates to possess immune-regulatory activities. Finally, we point out gaps in the molecular-level knowledge of this infectious agent that hinder our understanding of its immunology.
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Affiliation(s)
- A Díaz
- Cátedra de Inmunología, Departamento de Biociencias (Facultad de Química) and Instituto de Química Biológica (Facultad de Ciencias), Universidad de la República, Montevideo, Uruguay
| | - C Casaravilla
- Cátedra de Inmunología, Departamento de Biociencias (Facultad de Química) and Instituto de Química Biológica (Facultad de Ciencias), Universidad de la República, Montevideo, Uruguay
| | - A A Barrios
- Cátedra de Inmunología, Departamento de Biociencias (Facultad de Química) and Instituto de Química Biológica (Facultad de Ciencias), Universidad de la República, Montevideo, Uruguay
| | - A M Ferreira
- Cátedra de Inmunología, Departamento de Biociencias (Facultad de Química) and Instituto de Química Biológica (Facultad de Ciencias), Universidad de la República, Montevideo, Uruguay
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24
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Silva-Álvarez V, Folle AM, Ramos AL, Kitano ES, Iwai LK, Corraliza I, Córsico B, Ferreira AM. Echinococcus granulosus Antigen B binds to monocytes and macrophages modulating cell response to inflammation. Parasit Vectors 2016; 9:69. [PMID: 26846700 PMCID: PMC4743400 DOI: 10.1186/s13071-016-1350-7] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Accepted: 01/28/2016] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Antigen B (EgAgB) is an abundant lipoprotein released by the larva of the cestode Echinococcus granulosus into the host tissues. Its protein moiety belongs to the cestode-specific family known as hydrophobic ligand binding protein (HLBP), and is encoded by five gene subfamilies (EgAgB8/1-EgAgB8/5). The functions of EgAgB in parasite biology remain unclear. It may play a role in the parasite's lipid metabolism since it carries host lipids that E. granulosus is unable to synthesise. On the other hand, there is evidence supporting immuno-modulating activities in EgAgB, particularly on innate immune cells. Both hypothetical functions might involve EgAgB interactions with monocytes and macrophages, which have not been formally analysed yet. METHODS EgAgB binding to monocytes and macrophages was studied by flow cytometry using inflammation-recruited peritoneal cells and the THP-1 cell line. Involvement of the protein and phospholipid moieties in EgAgB binding to cells was analysed employing lipid-free recombinant EgAgB subunits and phospholipase D treated-EgAgB (lacking the polar head of phospholipids). Competition binding assays with plasma lipoproteins and ligands for lipoprotein receptors were performed to gain information about the putative EgAgB receptor(s) in these cells. Arginase-I induction and PMA/LPS-triggered IL-1β, TNF-α and IL-10 secretion were examined to investigate the outcome of EgAgB binding on macrophage response. RESULTS Monocytes and macrophages bound native EgAgB specifically; this binding was also found with lipid-free rEgAgB8/1 and rEgAgB8/3, but not rEgAgB8/2 subunits. EgAgB phospholipase D-treatment, but not the competition with phospholipid vesicles, caused a strong inhibition of EgAgB binding activity, suggesting an indirect contribution of phospholipids to EgAgB-cell interaction. Furthermore, competition binding assays indicated that this interaction may involve receptors with affinity for plasma lipoproteins. At functional level, the exposure of macrophages to EgAgB induced a very modest arginase-I response and inhibited PMA/LPS-mediated IL-1β and TNF-α secretion in an IL-10-independent manner. CONCLUSION EgAgB and, particularly its predominant EgAgB8/1 apolipoprotein, are potential ligands for monocyte and macrophage receptors. These receptors may also be involved in plasma lipoprotein recognition and induce an anti-inflammatory phenotype in macrophages upon recognition of EgAgB.
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Affiliation(s)
- Valeria Silva-Álvarez
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay. .,Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina. .,Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina.
| | - Ana Maite Folle
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay.
| | - Ana Lía Ramos
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay.
| | - Eduardo S Kitano
- Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signalling (CeTICS), Instituto Butantan, São Paulo, Brazil.
| | - Leo K Iwai
- Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signalling (CeTICS), Instituto Butantan, São Paulo, Brazil.
| | - Inés Corraliza
- Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura (UNEX), Cáceres, España.
| | - Betina Córsico
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina. .,Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina.
| | - Ana María Ferreira
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay.
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25
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An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis. J Clin Microbiol 2015; 53:3310-7. [PMID: 26269620 DOI: 10.1128/jcm.01362-15] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2015] [Accepted: 08/02/2015] [Indexed: 11/20/2022] Open
Abstract
Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.
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Silva-Álvarez V, Franchini GR, Pórfido JL, Kennedy MW, Ferreira AM, Córsico B. Lipid-free antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties. PLoS Negl Trop Dis 2015; 9:e0003552. [PMID: 25768648 PMCID: PMC4358968 DOI: 10.1371/journal.pntd.0003552] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2014] [Accepted: 01/21/2015] [Indexed: 02/06/2023] Open
Abstract
Background The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. Methodology/Principal Findings Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. Conclusions/Significance We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy. Echinococcus granulosus is a causative agent of hydatidosis, a parasitic disease that affects humans and livestock with significant economic and public health impact worldwide. Antigen B (EgAgB), an abundant product of E. granulosus larvae, is a lipoprotein that carries a wide variety of lipids, including fatty acids and cholesterol. As E. granulosus is unable to synthesize these lipids, EgAgB likely plays an important role in parasite metabolism, participating in both the acquisition of host lipids and their distribution between parasite tissues. The protein component of EgAgB consists of 8 kDa subunits encoded by separate genes. However, the biochemical properties of EgAgB subunits, particularly their ability to bind and transfer lipids, are poorly known. Herein, using in vitro assays, we found that EgAgB subunits were capable of oligomerizing in the absence of lipids and to bind fatty acids, but not cholesterol. Moreover, EgAgB subunits showed the ability to transfer fatty acids to artificial phospholipid membranes. These results indicate new points of attack at which the parasite might be vulnerable to drugs.
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Affiliation(s)
- Valeria Silva-Álvarez
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) (UNLP-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina
| | - Gisela R. Franchini
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) (UNLP-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina
| | - Jorge L. Pórfido
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) (UNLP-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina
| | - Malcolm W. Kennedy
- Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom
| | - Ana M. Ferreira
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Betina Córsico
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP) (UNLP-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina
- Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina
- * E-mail:
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Silva-Álvarez V, Folle AM, Ramos AL, Zamarreño F, Costabel MD, García-Zepeda E, Salinas G, Córsico B, Ferreira AM. Echinococcus granulosus antigen B: a Hydrophobic Ligand Binding Protein at the host-parasite interface. Prostaglandins Leukot Essent Fatty Acids 2015; 93:17-23. [PMID: 25451555 DOI: 10.1016/j.plefa.2014.09.008] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/15/2014] [Revised: 09/17/2014] [Accepted: 09/18/2014] [Indexed: 11/25/2022]
Abstract
Lipids are mainly solubilized by various families of lipid binding proteins which participate in their transport between tissues as well as cell compartments. Among these families, Hydrophobic Ligand Binding Proteins (HLBPs) deserve special consideration since they comprise intracellular and extracellular members, are able to bind a variety of fatty acids, retinoids and some sterols, and are present exclusively in cestodes. Since these parasites have lost catabolic and biosynthetic pathways for fatty acids and cholesterol, HLBPs are likely relevant for lipid uptake and transportation between parasite and host cells. Echinococcus granulosus antigen B (EgAgB) is a lipoprotein belonging to the HLBP family, which is very abundant in the larval stage of this parasite. Herein, we review the literature on EgAgB composition, structural organization and biological properties, and propose an integrated scenario in which this parasite HLBP contributes to adaptation to mammalian hosts by meeting both metabolic and immunomodulatory parasite demands.
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Affiliation(s)
- Valeria Silva-Álvarez
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad Nacional de La Plata (UNLP), La Plata, Argentina; Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Ana Maite Folle
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Ana Lía Ramos
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Fernando Zamarreño
- Grupo de Biofísica, Departamento de Física, Universidad Nacional del Sur (UNS), Bahía Blanca, Argentina
| | - Marcelo D Costabel
- Grupo de Biofísica, Departamento de Física, Universidad Nacional del Sur (UNS), Bahía Blanca, Argentina
| | - Eduardo García-Zepeda
- Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, Mexico
| | - Gustavo Salinas
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Betina Córsico
- Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad Nacional de La Plata (UNLP), La Plata, Argentina
| | - Ana María Ferreira
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay.
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Ahn CS, Han X, Bae YA, Ma X, Kim JT, Cai H, Yang HJ, Kang I, Wang H, Kong Y. Alteration of immunoproteome profile of Echinococcus granulosus hydatid fluid with progression of cystic echinococcosis. Parasit Vectors 2015; 8:10. [PMID: 25566682 PMCID: PMC4311513 DOI: 10.1186/s13071-014-0610-7] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Accepted: 12/17/2014] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed. METHODS We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages. RESULTS We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis. CONCLUSIONS Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.
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Affiliation(s)
- Chun-Seob Ahn
- Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Institute, Suwon, 440-746, Korea.
| | - Xiumin Han
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Xining, Qinghai, China.
| | - Young-An Bae
- Department of Microbiology, Graduate School of Medicine, Gachon University, Incheon, Korea.
| | - Xiao Ma
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Xining, Qinghai, China.
| | - Jin-Taek Kim
- Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Institute, Suwon, 440-746, Korea.
| | - Huixia Cai
- Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Institute, Suwon, 440-746, Korea.
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Xining, Qinghai, China.
| | - Hyun-Jong Yang
- Department of Parasitology, Ewha Womans University, School of Medicine, Seoul, Korea.
| | - Insug Kang
- Department of Molecular Biology and Biochemistry, School of Medicine, Kyung Hee University, Seoul, Korea.
| | - Hu Wang
- Qinghai Province Institute for Endemic Diseases Prevention and Control, Xining, Qinghai, China.
| | - Yoon Kong
- Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Institute, Suwon, 440-746, Korea.
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Pan W, Shen Y, Han X, Wang Y, Liu H, Jiang Y, Zhang Y, Wang Y, Xu Y, Cao J. Transcriptome profiles of the protoscoleces of Echinococcus granulosus reveal that excretory-secretory products are essential to metabolic adaptation. PLoS Negl Trop Dis 2014; 8:e3392. [PMID: 25500817 PMCID: PMC4263413 DOI: 10.1371/journal.pntd.0003392] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2014] [Accepted: 11/03/2014] [Indexed: 12/31/2022] Open
Abstract
Background Cystic hydatid disease (CHD) is caused by the larval stages of the cestode and affects humans and domestic animals worldwide. Protoscoleces (PSCs) are one component of the larval stages that can interact with both definitive and intermediate hosts. Previous genomic and transcriptomic data have provided an overall snapshot of the genomics of the growth and development of this parasite. However, our understanding of how PSCs subvert the immune response of hosts and maintains metabolic adaptation remains unclear. In this study, we used Roche 454 sequencing technology and in silico secretome analysis to explore the transcriptome profiles of the PSCs from E. granulosus and elucidate the potential functions of the excretory-secretory proteins (ESPs) released by the parasite. Methodology/Principal Findings A large number of nonredundant sequences as unigenes were generated (26,514), of which 22,910 (86.4%) were mapped to the newly published E. granulosus genome and 17,705 (66.8%) were distributed within the coding sequence (CDS) regions. Of the 2,280 ESPs predicted from the transcriptome, 138 ESPs were inferred to be involved in the metabolism of carbohydrates, while 124 ESPs were inferred to be involved in the metabolism of protein. Eleven ESPs were identified as intracellular enzymes that regulate glycolysis/gluconeogenesis (GL/GN) pathways, while a further 44 antigenic proteins, 25 molecular chaperones and four proteases were highly represented. Many proteins were also found to be significantly enriched in development-related signaling pathways, such as the TGF-β receptor pathways and insulin pathways. Conclusions/Significance This study provides valuable information on the metabolic adaptation of parasites to their hosts that can be used to aid the development of novel intervention targets for hydatid treatment and control. The successful infection establishment of parasites depends on their ability to combat their host's immune system while maintaining metabolic adaptation to their hosts. The mechanisms of these processes are not well understood. We used the protoscoleces (PSCs) of E. granulosus as a model system to study this complex host-parasite interaction by investigating the role of excretory-secretory proteins (ESPs) in the physiological adaptation of the parasite. Using Roche 454 sequencing technology and in silico secretome analysis, we predicted 2280 ESPs and analyzed their biological functions. Our analysis of the bioinformatic data suggested that ESPs are integral to the metabolism of carbohydrates and proteins within the parasite and/or hosts. We also found that ESPs are involved in mediating the immune responses of hosts and function within key development-related signaling pathways. We found 11 intracellular enzymes, 25 molecular chaperones and four proteases that were highly represented in the ESPs, in addition to 44 antigenic proteins that showed promise as candidates for vaccine or serodiagnostic development purposes. These findings provide valuable information on the mechanisms of metabolic adaptation in parasites that will aid the development of novel hydatid treatment and control targets.
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Affiliation(s)
- Wei Pan
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Yujuan Shen
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
- * E-mail: (YS); (JC)
| | - Xiuming Han
- Department of Parasitic Diseases, Qinghai Institute for Endemic Disease Prevention and Control, Zong Zhai, Xining, Qinghai, People's Republic of China
| | - Ying Wang
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Hua Liu
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Yanyan Jiang
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Yumei Zhang
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Yanjuan Wang
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Yuxin Xu
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
| | - Jianping Cao
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China
- Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, People's Republic of China
- WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, People's Republic of China
- * E-mail: (YS); (JC)
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Bai Y, Zhang Z, Jin L, Kang H, Zhu Y, Zhang L, Li X, Ma F, Zhao L, Shi B, Li J, McManus DP, Zhang W, Wang S. Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus. BMC Genomics 2014; 15:736. [PMID: 25168356 PMCID: PMC4156656 DOI: 10.1186/1471-2164-15-736] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Accepted: 08/20/2014] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND MicroRNAs (miRNAs) are important post-transcriptional regulators which control growth and development in eukaryotes. The cestode Echinococcus granulosus has a complex life-cycle involving different development stages but the mechanisms underpinning this development, including the involvement of miRNAs, remain unknown. RESULTS Using Illumina next generation sequencing technology, we sequenced at the genome-wide level three small RNA populations from the adult, protoscolex and cyst membrane of E. granulosus. A total of 94 pre-miRNA candidates (coding 91 mature miRNAs and 39 miRNA stars) were in silico predicted. Through comparison of expression profiles, we found 42 mature miRNAs and 23 miRNA stars expressed with different patterns in the three life stages examined. Furthermore, considering both the previously reported and newly predicted miRNAs, 25 conserved miRNAs families were identified in the E. granulosus genome. Comparing the presence or absence of these miRNA families with the free-living Schmidtea mediterranea, we found 13 conserved miRNAs are lost in E. granulosus, most of which are tissue-specific and involved in the development of ciliated cells, the gut and sensory organs. Finally, GO enrichment analysis of the differentially expressed miRNAs and their potential targets indicated that they may be involved in bi-directional development, nutrient metabolism and nervous system development in E. granulosus. CONCLUSIONS This study has, for the first time, provided a comprehensive description of the different expression patterns of miRNAs in three distinct life cycle stages of E. granulosus. The analysis supports earlier suggestions that the loss of miRNAs in the Platyhelminths might be related to morphological simplification. These results may help in the exploration of the mechanism of interaction between this parasitic worm and its definitive and intermediate hosts, providing information that can be used to develop new interventions and therapeutics for the control of cystic echinococcosis.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | | | - Wenbao Zhang
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, 250 Bibo Road, Shanghai 201203, China.
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Espínola SM, Ferreira HB, Zaha A. Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages. PLoS One 2014; 9:e102228. [PMID: 25014071 PMCID: PMC4094502 DOI: 10.1371/journal.pone.0102228] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Accepted: 06/16/2014] [Indexed: 12/02/2022] Open
Abstract
In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.
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Affiliation(s)
- Sergio Martin Espínola
- Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Henrique Bunselmeyer Ferreira
- Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências, and Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Arnaldo Zaha
- Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências, and Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- * E-mail:
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McManus DP. Immunodiagnosis of sheep infections withEchinococcus granulosus: in 35 years where have we come? Parasite Immunol 2014; 36:125-30. [DOI: 10.1111/pim.12072] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2013] [Accepted: 08/12/2013] [Indexed: 01/22/2023]
Affiliation(s)
- D. P. McManus
- Molecular Parasitology Laboratory; Division of Infectious Diseases; Queensland Institute of Medical Research; Herston Qld Australia
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Mariconti M, Bazzocchi C, Tamarozzi F, Meroni V, Genco F, Maserati R, Brunetti E. Immunoblotting with human native antigen shows stage-related sensitivity in the serodiagnosis of hepatic cystic echinococcosis. Am J Trop Med Hyg 2013; 90:75-9. [PMID: 24297816 DOI: 10.4269/ajtmh.13-0341] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The diagnosis of hepatic cystic echinococcosis is based on ultrasonography and confirmed by serology. However, no biological marker of cyst viability is currently available implying years-long patient follow-up, which is not always feasible in endemic areas. We characterized the performance of an immunoblotting test based on human hydatid cyst fluid with particular regard to its ability to distinguish between cyst stages. Sera from patients with cysts in different stages showed distinctive band pattern recognition. Most importantly, the test discriminated in 80% of cases CE3a from CE3b transitional cysts, known to have different viability profiles. Interestingly, we observed a rapid change in band pattern recognition of sera from one patient at time points when his cyst passed from active to transitional to inactive stages. Further identification of different antigens expressed by different cyst stages will support the development of diagnostic tools that could early define cyst viability, to guide clinical decision making, and shorten patient follow-up.
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Affiliation(s)
- Mara Mariconti
- Policlinico San Matteo Hospital Foundation, Pavia, Italy; Department of Veterinary Science and Public Health, University of Milan, Milan, Italy; Department of Clinical Surgical Diagnostic and Paediatric Sciences, University of Pavia, Pavia, Italy; World Health Organization Collaborating Centre for Clinical Management of Cystic Echinococcosis, Pavia, Italy
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SARKARI (SHAHRIARI) B, BIRANVAND E, SADJJADI SM, RAHIMI H. Genetic Variability of Antigen B2 of Human, Sheep, Goats, Camel and Cattle Isolates of Echinococcus granulosus in Iran. IRANIAN JOURNAL OF PARASITOLOGY 2013; 8:545-551. [PMID: 25516735 PMCID: PMC4266118] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/10/2013] [Accepted: 07/19/2013] [Indexed: 10/29/2022]
Abstract
BACKGROUND Antigen B (AgB) is frequently used for immuno-diagnosis of human cystic echinococcosis (CE). Echinococcus granulosus AgBs show a high degree of genetic variability in different hosts or in different CE endemic areas. The present study aimed to evaluate the genetic polymorphisms of encoding antigen B2 gene (AgB2) among different Iranian isolates of E. granulosus. METHODS A total of 50 CE isolates were collected from human, sheep, cattle, goat and camels, 10 isolates from each intermediate host of E. granulosus. Total genomic DNA from either protoscolices or germinal layer was extracted from each cyst and PCR-RFLP followed by DNA sequencing was used to evaluate sequence variation and polymorphism of AgB2 in the isolates. RESULTS After the PCR amplification, using AgB2 primers, an almost 400 bp band was amplified in all of the isolates. The PCR products were digested with Alu1 restriction endonuclease. After restriction enzyme digestion with Alu1' sheep and human isolates gave a similar pattern of RFLP with the gene size of approximately 140 and 240bp and camel and goat isolates gave a similar pattern, but different from sheep and human, with the gene size of approximately 150 and 250bp. Sequence analysis showed the most genetic similarity of AgB2 between human and sheep isolates. CONCLUSION Findings of this study revealed the differences in the sequences of AgB2 within and between the Iranian isolates of E. granulosus. These differences may affect the performance of any diagnostic test which uses AgB.
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Affiliation(s)
| | - Elahe BIRANVAND
- Dept. of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Seyed Mahmoud SADJJADI
- Dept. of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Hamidreza RAHIMI
- Dept. of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
- School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
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Barteneva NS, Maltsev N, Vorobjev IA. Microvesicles and intercellular communication in the context of parasitism. Front Cell Infect Microbiol 2013; 3:49. [PMID: 24032108 PMCID: PMC3764926 DOI: 10.3389/fcimb.2013.00049] [Citation(s) in RCA: 77] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2013] [Accepted: 08/20/2013] [Indexed: 01/18/2023] Open
Abstract
There is a rapidly growing body of evidence that production of microvesicles (MVs) is a universal feature of cellular life. MVs can incorporate microRNA (miRNA), mRNA, mtDNA, DNA and retrotransposons, camouflage viruses/viral components from immune surveillance, and transfer cargo between cells. These properties make MVs an essential player in intercellular communication. Increasing evidence supports the notion that MVs can also act as long-distance vehicles for RNA molecules and participate in metabolic synchronization and reprogramming eukaryotic cells including stem and germinal cells. MV ability to carry on DNA and their general distribution makes them attractive candidates for horizontal gene transfer, particularly between multi-cellular organisms and their parasites; this suggests important implications for the co-evolution of parasites and their hosts. In this review, we provide current understanding of the roles played by MVs in intracellular pathogens and parasitic infections. We also discuss the possible role of MVs in co-infection and host shifting.
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Affiliation(s)
- Natasha S Barteneva
- Program in Cellular and Molecular Medicine, Children's Hospital Boston and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Natasha.Barteneva@ childrens.harvard.edu
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Zheng Y. Strategies of Echinococcus species responses to immune attacks: implications for therapeutic tool development. Int Immunopharmacol 2013; 17:495-501. [PMID: 23973651 DOI: 10.1016/j.intimp.2013.07.022] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2013] [Revised: 07/26/2013] [Accepted: 07/30/2013] [Indexed: 01/27/2023]
Abstract
Echinococcus species have been studied as a model to investigate parasite-host interactions. Echinococcus spp. can actively communicate dynamically with a host to facilitate infection, growth and proliferation partially via secretion of molecules, especially in terms of harmonization of host immune attacks. This review systematically outlines our current knowledge of how the Echinococcus species have evolved to adapt to their host's microenvironment. This understanding of parasite-host interplay has implications in profound appreciation of parasite plasticity and is informative in designing novel and effective tools including vaccines and drugs for the treatment of echinococcosis and other diseases.
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Affiliation(s)
- Yadong Zheng
- State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu, China; Key Lab of New Animal Drug Project, Gansu Province, Lanzhou Institute of Husbandry, Pharmaceutical Sciences, CAAS, Lanzhou, Gansu, China; Key Lab of Veterinary Pharmaceutical Development, Ministry of Agriculture, Lanzhou Institute of Husbandry, Pharmaceutical Sciences, CAAS, Lanzhou, Gansu, China.
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Haçarız O, Sayers G. Fasciola hepatica - where is 28S ribosomal RNA? Exp Parasitol 2013; 135:426-9. [PMID: 23954260 DOI: 10.1016/j.exppara.2013.07.026] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2013] [Accepted: 07/31/2013] [Indexed: 11/26/2022]
Abstract
Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.
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Affiliation(s)
- Orçun Haçarız
- TÜBİTAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze, Kocaeli, Turkey.
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Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient's sera: a novel hypothesis to follow-up of Cystic echinococcosis after surgery. Med Hypotheses 2013; 81:557-60. [PMID: 23890801 DOI: 10.1016/j.mehy.2013.07.003] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2013] [Revised: 06/24/2013] [Accepted: 07/02/2013] [Indexed: 11/23/2022]
Abstract
Cystic echinococcosis (CE) is caused by the larval stage of the tapeworm Echinococcus granulosus. Until now, CE does not have an effective follow-up after surgery. To date, CE follow-up is conducted based on either antibody or antigen detection assays by double antibody sandwich ELISA. Unlike antigen detection, antibodies to imply exposure to an Echinococcus infection while their titration could remain for a longer period (1-10years) after surgery. Likewise, antibody respond may be related to the location of a mature hydatid cyst. Antigen detection shows presence of CE infection, it is extremely important and necessary in follow-up of CE after surgery. The circulating antigen (CAg) titration decrease faster than circulating antibody (during 1-3weeks) after operation. Location of hydatid cyst in detecting antigen is affected less than antibody also. Regarding this subject, antigen detection has several limitations that lead to be used less in CE follow-up. Although, AgB 8/1-kDa subunit is considered as a principle and immunogenic CAg but sensitivity of CAg detection compared to with antibody has variable range, between 33% and 85% which owing to formation of circulating immune complexes (CICs) in result of antigen - antibody complex. The another problem is non using specific CAg (AgB 8/1-kDa subunit) for production of specific paratopes (rabbit hyper immune antiserum) against AgB 8/1-kDa which is used as capture (primary) antibody in double antibody sandwich ELISA assay. The designation of synthetic peptides from conserved regions of AgB 8/1-kDa can help to this problem. These regions (motifs) should be selected for allelic, dominant, immunogenic and conserved without any genetic variation. The first part of this hypothesis suggests which patient's sera should be treated with acidic buffers such as boric acid, acetic acid, glycine/HCl, polyethylene glycol (PEG) or combination of each of them accompanied by boiling patient's sera which causes breaking Ag-Ab complexes and in result of releasing AgB 8/1. These modifications are effective to releasing CAg from CIC. To date, the synthetic peptides have been widely used in CE serodiagnosis based on circulating antibody detection only. In second part of this hypothesis suggests the using synthetic peptide of p176 derived AgB 8/1-kDa subunit containing conserved specific epitopes for preparation of specific paratopes (rabbit hyper immune antiserum) based on CAg detection. So, there is no need any native antigens for preparation of non-specific rabbit hyper immune antiserum. These novel improvements can help to decrease the cross-reactivity with other parasitic diseases (specificity). Increasing antigen detection could make a chance in sensitivity of patient's sera and in result of the best and suitable tool for CE follow-up.
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Cui SJ, Xu LL, Zhang T, Xu M, Yao J, Fang CY, Feng Z, Yang PY, Hu W, Liu F. Proteomic characterization of larval and adult developmental stages in Echinococcus granulosus reveals novel insight into host-parasite interactions. J Proteomics 2013; 84:158-75. [PMID: 23603110 DOI: 10.1016/j.jprot.2013.04.013] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2012] [Revised: 04/08/2013] [Accepted: 04/09/2013] [Indexed: 10/26/2022]
Abstract
UNLABELLED Cystic hydatid disease is an important zoonosis caused by Echinococcus granulosus infection. The expression profiles of its parasitic life stages and host-Echinococcus interactions remain to be elucidated. Here, we identified 157 adult and 1588 protoscolex proteins (1610 in all), including 1290 novel identifications. Paramyosins and an antigen B (AgB) were the dominant adult proteins. Dog proteins (30) identified in adults indicated diminished local inflammation caused by adult infection. The protoscolex expresses proteins that have been reported to be antigens in other parasites, such as 6-phosphofructokinase and calcineurin B. Pathway analyses suggested that E. granulosus uses both aerobic and anaerobic carbohydrate metabolisms to generate ATP. E. granulosus expresses proteins involved in synthesis and metabolism of lipids or steroids. At least 339 of 390 sheep proteins identified in protoscolex were novel identifications not seen in previous analyses. IgGs and lambda light chains were the most abundant antibody species. Sheep proteins were enriched for detoxification pathways, implying that host detoxification effects play a central role during host-parasite interactions. Our study provides valuable data on E. granulosus expression characteristics, allowing novel insights into the molecular mechanisms involved in host-parasite interactions. BIOLOGICAL SIGNIFICANCE In this study, the Echinococcus granulosus adult worm proteome was analyzed for the first time. The protein identification of E. granulosus protoscoleces was extended dramatically. We also identified the most abundant host proteins co-purified with Echinococcus. The results provide useful information pertaining to the molecular mechanisms behind host-Echinococcus interaction and Echinococcus biology. This data also increases the potential for identifying vaccine candidates and new therapeutic targets, and may aid in the development of protein probes for selective and sensitive diagnosis of echinococcosis infection. In addition, the data collected here represents a valuable proteomic resource for subsequent genome annotation.
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Affiliation(s)
- Shu-Jian Cui
- Institutes of Biomedical Sciences, Fudan University, 131 Dongan Road, Shanghai 200032, China
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Parkinson J, Wasmuth JD, Salinas G, Bizarro CV, Sanford C, Berriman M, Ferreira HB, Zaha A, Blaxter ML, Maizels RM, Fernández C. A transcriptomic analysis of Echinococcus granulosus larval stages: implications for parasite biology and host adaptation. PLoS Negl Trop Dis 2012; 6:e1897. [PMID: 23209850 PMCID: PMC3510090 DOI: 10.1371/journal.pntd.0001897] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2012] [Accepted: 09/25/2012] [Indexed: 01/14/2023] Open
Abstract
Background The cestode Echinococcus granulosus - the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide - is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages. Methodology/Principal Findings We generated ∼10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H+-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development. Conclusions/Significance This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths. Cestodes are a neglected group of platyhelminth parasites, despite causing chronic infections to humans and domestic animals worldwide. We used Echinococcus granulosus as a model to study the molecular basis of the host-parasite cross-talk during cestode infections. For this purpose, we carried out a survey of the genes expressed by parasite larval stages interfacing with definitive and intermediate hosts. Sequencing from several high quality cDNA libraries provided numerous insights into the expression of genes involved in important aspects of E. granulosus biology, e.g. its metabolism (energy production and antioxidant defences) and the synthesis of key parasite structures (notably, the one exposed to humans and livestock intermediate hosts). Our results also uncovered the existence of an intriguing set of abundant repeat-associated non-protein coding transcripts that may participate in the regulation of gene expression in all surveyed stages. The dataset now generated constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic studies focused on cestodes and platyhelminths. In particular, the detailed characterization of a range of newly discovered genes will contribute to a better understanding of the biology of cestode infections and, therefore, to the development of products allowing their efficient control.
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Affiliation(s)
- John Parkinson
- Program in Molecular Structure and Function, Hospital for Sick Children, University of Toronto, Toronto, Canada
| | - James D. Wasmuth
- Program in Molecular Structure and Function, Hospital for Sick Children, University of Toronto, Toronto, Canada
| | - Gustavo Salinas
- Cátedra de Inmunología, Facultad de Química, Universidad de la República, Montevideo, Uruguay
| | - Cristiano V. Bizarro
- Laboratório de Biologia Molecular de Cestódeos and Laboratorio de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - Chris Sanford
- Program in Molecular Structure and Function, Hospital for Sick Children, University of Toronto, Toronto, Canada
| | - Matthew Berriman
- Parasite Genomics, The Wellcome Trust Sanger Institute, Hinxton, United Kingdom
| | - Henrique B. Ferreira
- Laboratório de Biologia Molecular de Cestódeos and Laboratorio de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - Arnaldo Zaha
- Laboratório de Biologia Molecular de Cestódeos and Laboratorio de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - Mark L. Blaxter
- Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom
| | - Rick M. Maizels
- Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom
- * E-mail: (RMM); (CF)
| | - Cecilia Fernández
- Cátedra de Inmunología, Facultad de Química, Universidad de la República, Montevideo, Uruguay
- * E-mail: (RMM); (CF)
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Olson PD, Zarowiecki M, Kiss F, Brehm K. Cestode genomics - progress and prospects for advancing basic and applied aspects of flatworm biology. Parasite Immunol 2012; 34:130-50. [PMID: 21793855 DOI: 10.1111/j.1365-3024.2011.01319.x] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Characterization of the first tapeworm genome, Echinococcus multilocularis, is now nearly complete, and genome assemblies of E. granulosus, Taenia solium and Hymenolepis microstoma are in advanced draft versions. These initiatives herald the beginning of a genomic era in cestodology and underpin a diverse set of research agendas targeting both basic and applied aspects of tapeworm biology. We discuss the progress in the genomics of these species, provide insights into the presence and composition of immunologically relevant gene families, including the antigen B- and EG95/45W families, and discuss chemogenomic approaches toward the development of novel chemotherapeutics against cestode diseases. In addition, we discuss the evolution of tapeworm parasites and introduce the research programmes linked to genome initiatives that are aimed at understanding signalling systems involved in basic host-parasite interactions and morphogenesis.
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Affiliation(s)
- P D Olson
- Department of Zoology, The Natural History Museum, London, UK
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Characterization of hydrophobic-ligand-binding proteins of Taenia solium that are expressed specifically in the adult stage. Parasitology 2012; 139:1361-74. [PMID: 22657393 DOI: 10.1017/s0031182012000613] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Taenia solium, a causative agent of taeniasis and cysticercosis, has evolved a repertoire of lipid uptake mechanisms. Proteome analysis of T. solium excretory-secretory products (TsESP) identified 10 kDa proteins displaying significant sequence identity with cestode hydrophobic-ligand-binding-proteins (HLBPs). Two distinct 362- and 352-bp-long cDNAs encoding 264- and 258-bp-long open reading frames (87 and 85 amino acid polypeptides) were isolated by mining the T. solium expressed sequence tags and a cDNA library screening (TsHLBP1 and TsHLBP2; 94% sequence identity). They clustered into the same clade with those found in Moniezia expansa and Hymenolepis diminuta. Genomic structure analysis revealed that these genes might have originated from a common ancestor. Both the crude TsESP and bacterially expressed recombinant proteins exhibited binding activity toward 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), which was competitively inhibited by oleic acid. The proteins also bound to cis-parinaric acid (cPnA) and 16-(9-anthroyloxy) palmitic acid (16-AP), but showed no binding activity against 11-[(5-dimethylaminonaphthalene-1-sulfonyl) amino] undecanoic acid (DAUDA) and dansyl-DL-α-aminocaprylic acid (DACA). Unsaturated fatty acids (FAs) showed greater affinity than saturated FAs. The proteins were specifically expressed in adult worms throughout the strobila. The TsHLBPs might be involved in uptake and/or sequestration of hydrophobic molecules provided by their hosts, thus contributing to host-parasite interface interrelationships.
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Obal G, Ramos AL, Silva V, Lima A, Batthyany C, Bessio MI, Ferreira F, Salinas G, Ferreira AM. Characterisation of the native lipid moiety of Echinococcus granulosus antigen B. PLoS Negl Trop Dis 2012; 6:e1642. [PMID: 22616019 PMCID: PMC3352830 DOI: 10.1371/journal.pntd.0001642] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2012] [Accepted: 03/29/2012] [Indexed: 12/21/2022] Open
Abstract
Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.
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Affiliation(s)
- Gonzalo Obal
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
- Unidad de Biofísica de Proteínas, Instituto Pasteur de Montevideo, Montevideo, Uruguay
| | - Ana Lía Ramos
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Valeria Silva
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Analía Lima
- Unidad de Bioquímica y Proteómica Analíticas, Instituto Pasteur de Montevideo, Montevideo, Uruguay
| | - Carlos Batthyany
- Unidad de Bioquímica y Proteómica Analíticas, Instituto Pasteur de Montevideo, Montevideo, Uruguay
| | - María Inés Bessio
- Laboratorio de Carbohidratos y Glicoconjugados, Departamento de Química Orgánica/Departamento de Desarrollo Biotecnológico, Facultad de Química/Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Fernando Ferreira
- Laboratorio de Carbohidratos y Glicoconjugados, Departamento de Química Orgánica/Departamento de Desarrollo Biotecnológico, Facultad de Química/Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Gustavo Salinas
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
| | - Ana María Ferreira
- Cátedra de Inmunología, Facultad de Ciencias/Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay
- * E-mail:
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Monteiro KM, Cardoso MB, Follmer C, da Silveira NP, Vargas DM, Kitajima EW, Zaha A, Ferreira HB. Echinococcus granulosus antigen B structure: subunit composition and oligomeric states. PLoS Negl Trop Dis 2012; 6:e1551. [PMID: 22413028 PMCID: PMC3295803 DOI: 10.1371/journal.pntd.0001551] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2011] [Accepted: 01/12/2012] [Indexed: 12/28/2022] Open
Abstract
BACKGROUND Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. METHODOLOGY/PRINCIPAL FINDINGS The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. CONCLUSIONS/SIGNIFICANCE For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.
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Affiliation(s)
- Karina M. Monteiro
- Laboratório de Biologia Molecular de Cestódeos and Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Mateus B. Cardoso
- Laboratório Nacional de Luz Síncrotron (LNLS), Campinas, São Paulo, Brazil
| | - Cristian Follmer
- Departamento de Físico-Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil
| | - Nádya P. da Silveira
- Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Daiani M. Vargas
- Laboratório de Biologia Molecular de Cestódeos and Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Elliot W. Kitajima
- Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz (ESALQ), Universidade de São Paulo, Piracicaba, São Paulo, Brazil
| | - Arnaldo Zaha
- Laboratório de Biologia Molecular de Cestódeos and Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Henrique B. Ferreira
- Laboratório de Biologia Molecular de Cestódeos and Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
- * E-mail:
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Nono JK, Pletinckx K, Lutz MB, Brehm K. Excretory/secretory-products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro. PLoS Negl Trop Dis 2012; 6:e1516. [PMID: 22363826 PMCID: PMC3283565 DOI: 10.1371/journal.pntd.0001516] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2011] [Accepted: 12/19/2011] [Indexed: 12/19/2022] Open
Abstract
BACKGROUND Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. METHODOLOGY/PRINCIPAL FINDINGS We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. CONCLUSIONS This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.
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Affiliation(s)
- Justin Komguep Nono
- University of Würzburg, Institute of Hygiene and Microbiology, Würzburg, Germany
| | - Katrien Pletinckx
- University of Würzburg, Institute of Virology and Immunobiology, Würzburg, Germany
| | - Manfred B. Lutz
- University of Würzburg, Institute of Virology and Immunobiology, Würzburg, Germany
| | - Klaus Brehm
- University of Würzburg, Institute of Hygiene and Microbiology, Würzburg, Germany
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Abstract
Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.
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Virginio VG, Monteiro KM, Drumond F, de Carvalho MO, Vargas DM, Zaha A, Ferreira HB. Excretory/secretory products from in vitro-cultured Echinococcus granulosus protoscoleces. Mol Biochem Parasitol 2012; 183:15-22. [PMID: 22261090 DOI: 10.1016/j.molbiopara.2012.01.001] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2011] [Revised: 12/22/2011] [Accepted: 01/04/2012] [Indexed: 01/07/2023]
Abstract
Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host-parasite relationship. In this work, we performed an LC-MS/MS proteomic analysis of the excretory-secretory products obtained from the first 48 h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory-secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory-secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory-secretory products, revealing a set of identified proteins not previously thought to be exposed at the host-parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory-secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory-secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.
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Affiliation(s)
- Veridiana G Virginio
- Laboratório de Biologia Molecular de Cestódeos e Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil
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Siracusano A, Delunardo F, Teggi A, Ortona E. Host-parasite relationship in cystic echinococcosis: an evolving story. Clin Dev Immunol 2011; 2012:639362. [PMID: 22110535 PMCID: PMC3206507 DOI: 10.1155/2012/639362] [Citation(s) in RCA: 96] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2011] [Accepted: 09/27/2011] [Indexed: 01/29/2023]
Abstract
The larval stage of Echinococcus granulosus causes cystic echinococcosis, a neglected infectious disease that constitutes a major public health problem in developing countries. Despite being under constant barrage by the immune system, E. granulosus modulates antiparasite immune responses and persists in the human hosts with detectable humoral and cellular responses against the parasite. In vitro and in vivo immunological approaches, together with molecular biology and immunoproteomic technologies, provided us exciting insights into the mechanisms involved in the initiation of E. granulosus infection and the consequent induction and regulation of the immune response. Although the last decade has clarified many aspects of host-parasite relationship in human cystic echinococcosis, establishing the full mechanisms that cause the disease requires more studies. Here, we review some of the recent developments and discuss new avenues in this evolving story of E. granulosus infection in man.
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Affiliation(s)
- Alessandra Siracusano
- Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy.
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Kim SH, Bae YA, Yang Y, Hong ST, Kong Y. Paralogous proteins comprising the 150 kDa hydrophobic-ligand-binding-protein complex of the Taenia solium metacestode have evolved non-overlapped binding affinities toward fatty acid analogs. Int J Parasitol 2011; 41:1207-15. [PMID: 21839082 DOI: 10.1016/j.ijpara.2011.07.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Revised: 07/07/2011] [Accepted: 07/19/2011] [Indexed: 10/17/2022]
Abstract
We previously identified a hydrophobic-ligand-binding protein (HLBP) of the Taenia solium metacestode (TsM), which might be involved in the uptake of fatty acids (FAs) from host environments. The TsM 150kDa HLBP was a hetero-oligomeric complex composed of multiple 7kDa (RS1) and 10kDa (CyDA, b1 and m13h) subunits, and displayed a wide spectrum of binding affinities toward various FA analogs. In this study, we analysed biochemical properties and phylogenetic relationships of the individual subunits. Despite the low sequence identity (average 26.5%), these subunit proteins conserved an α-helix-rich structural domain and the first introns inserted in each of the respective chromosomal genes were found to be orthologous to one another, suggesting their common evolutionary origin. The recombinant RS1 protein bound strongly to all of the FA analogs examined including 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino]undecanoic acid (DAUDA), but not to 16-(9-anthroyloxy)palmitic acid (16-AP). The interactive binding between RS1 and FA analogs was specifically interfered with by the addition of non-fluorescent FA molecules or antibodies specific to the 150kDa protein. Conversely, the 10kDa members reacted only with the palmitic acid-derived 16-AP, whose interactive force was strengthened by the presence of other FA molecules. The use of mutagenic RS1 proteins demonstrated that a structural/electrostatic integrity around the second α-helix, rather than the conventional Trp residue, was the major factor governing the hydrophobic interaction. The 7 and 10kDa proteins exhibited distinctive immunoreactive patterns against sera from neurocysticercosis patients. These collective data suggest that the paralogous protein family have gained diverse functions during their evolution, to ensure the maintenance of metabolic homeostasis and survival of TsMs in hostile host environments.
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Affiliation(s)
- Seon-Hee Kim
- Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Center, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea
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