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.

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.

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Bioinformatic Comparison of Kunitz Protease Inhibitors in Echinococcus Granulosus and E. Multilocularis and the Genes Expressed in Different Developmental Stages of E. Granulosus

10.21203/rs.3.rs-318603/v1 ◽

2021 ◽

Author(s):

Hui Zhang ◽

Mengxiao Tian ◽

Wenjing Qi ◽

Juan Wu ◽

Huajun Zheng ◽

...

Keyword(s):

Protease Inhibitors ◽

Echinococcus Granulosus ◽

Alveolar Echinococcosis ◽

Developmental Stages ◽

Zoonotic Diseases ◽

Neighbor Joining ◽

Transcriptome Data ◽

Kunitz Domain ◽

Kunitz Type ◽

Echinococcus Spp

Abstract Background: Cystic and alveolar echinococcosis caused by the tapeworms Echinococcus granulosus 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 all the Kunitz-type protease inhibitors 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. Results: A total of 19 genes from E. multilocularis and 23 genes from E. granulosus contained single or multiple Kunitz-domains. A neighbor-joining phylogenetic tree indicated that the E. granulosus and E. multilocularis Kunitz domain peptides were divided into three branches containing 9 clusters. Based on available transcriptome data, we analyzed the expression of these Kunitz-domain protease inhibitors in four major developmental stages of E. granulosus and found they were differentially expressed.Conclusion: We identified 19 and 23 Kunitz protease inhibitors in E. multilocularis and E. granulosus respectively; the majority of these genes were expressed in one or four stages of E. granulosus with some being highly expressed in adult worms indicating that these genes likely play different roles in the different developmental stages.

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Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0004268 ◽

2015 ◽

Vol 9 (12) ◽

pp. e0004268 ◽

Cited By ~ 19

Author(s):

Shiwanthi L. Ranasinghe ◽

Katja Fischer ◽

Wenbao Zhang ◽

Geoffrey N. Gobert ◽

Donald P. McManus

Keyword(s):

Protease Inhibitors ◽

Echinococcus Granulosus ◽

Kunitz Type

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Identification and characterisation of novel Kunitz type protease inhibitors from Echinococcus granulosus, Schistosoma mansoni and Schiatoaom japonicum

10.14264/uql.2016.941 ◽

2016 ◽

Author(s):

Shiwanthi Ranasinghe

Keyword(s):

Schistosoma Mansoni ◽

Protease Inhibitors ◽

Echinococcus Granulosus ◽

Kunitz Type

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Response patterns in adventitial layer of Echinococcus granulosus sensu stricto cysts from naturally infected cattle and sheep

Veterinary Research ◽

10.1186/s13567-021-00936-8 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Christian Hidalgo ◽

Caroll Stoore ◽

María Soledad Baquedano ◽

Ismael Pereira ◽

Carmen Franco ◽

...

Keyword(s):

Immune Response ◽

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Direct Contact ◽

Sensu Stricto ◽

Response Patterns ◽

Granulomatous Reaction ◽

Adventitial Layer ◽

Laminated Layer ◽

Cattle And Sheep

AbstractCystic echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus granulosus sensu lato. The disease is characterized by the development of cystic structures inside viscera of the intermediate host, mainly liver and lungs. These cysts are formed by three layers: germinal, laminated, and adventitial layer, the latter being the local host immune response. Metacestodes that develop protoscoleces, the infective stage to the definitive host, are termed fertile, whereas cysts that do not produce protoscoleces are termed non-fertile. Sheep usually harbor fertile cysts while cattle usually harbor non-fertile cysts. Adventitial layers with fibrotic resolution are associated to fertile cysts, whereas a granulomatous reaction is associated with non-fertile cysts. The aim of this study was to analyze cellular distribution in the adventitial layer of fertile and non-fertile E. granulosus sensu stricto cysts found in liver and lungs of cattle and sheep. A total of 418 cysts were analyzed, 203 from cattle (8 fertile and 195 non-fertile) and 215 from sheep (64 fertile and 151 non-fertile). Fertile cysts from cattle showed mixed patterns of response, with fibrotic resolution and presence of granulomatous response in direct contact with the laminated layer, while sheep fertile cysts always displayed fibrotic resolution next to the laminated layer. Cattle non-fertile cysts display a granulomatous reaction in direct contact with the laminated layer, whereas sheep non-fertile cysts display a granulomatous reaction, but in direct contact with the fibrotic resolution. This shows that cattle and sheep cystic echinococcosis cysts have distinct local immune response patterns, which are associated to metacestode fertility.

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Human Cystic Echinococcosis in Lebanon: A Retrospective Study and Molecular Epidemiology

Acta Parasitologica ◽

10.1007/s11686-021-00453-w ◽

2021 ◽

Author(s):

Gaelle Joanny ◽

Maria Grazia Cappai ◽

Francesca Nonnis ◽

Claudia Tamponi ◽

Giorgia Dessì ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Epidemiological Data ◽

Sensu Stricto ◽

Control Measures ◽

Mediterranean Countries ◽

High Gene ◽

Human Infections ◽

And Control ◽

Human Cystic Echinococcosis

Abstract Purpose Human cystic echinococcosis (CE) is a zoonotic parasitic disease that constitutes a public health challenge and a socio-economic burden in endemic areas worldwide. No specific surveillance system of CE infections in humans exists in Lebanon. The incidence and trends over time have not been documented. The current study aimed to assess the demographic and epidemiologic features of human CE surgical cases over a 14-year period in the five main regions of Lebanon. Methods From 2005 to 2018, a total of 894 surgically confirmed cases of hydatidosis were recorded from five anatomy and pathology laboratories. Results The mean annual surgical incidence was 1.23/100,000 inhabitants. Over the span of these years, the incidence increased from 0.53 to 1.94 cases/100,000 inhabitants in 2005 and 2018, respectively. CE is present in Lebanon with an uneven distribution from one region to the other with higher prevalence in Bekaa (29.0%), a rural area where sheep raising is widespread. Human CE cases were more common in females (60.1%) than in males (39.9%) and a high burden of infection was reported for the age group of 30–39 years. Besides, 66.7% of the cases expressed only liver complications whereas, 20.5% showed predilection towards lungs. The 7.8% of cases presented cysts in other organs, and 1.3% showed multiple localizations. Additionally, predominant involvement of Echinococcus granulosus sensu stricto was recorded in human infections. Comparison of Echinococcus granulosus s.s. populations from different Mediterranean countries also revealed high gene flow among this region and sharing of alleles. Conclusion The current study is a step forward to fill the gap of knowledge for the hydatidosis in Lebanon where the lack of epidemiological data and control measures have resulted in higher incidence of human CE. Graphic Abstract

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Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays

Pathogens ◽

10.3390/pathogens10020125 ◽

2021 ◽

Vol 10 (2) ◽

pp. 125

Author(s):

Piero Bonelli ◽

Silvia Dei Giudici ◽

Angela Peruzzu ◽

Lorena Mura ◽

Cinzia Santucciu ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Animal Species ◽

Cost Effective ◽

Sensu Stricto ◽

Sybr Green ◽

Hydatid Cysts ◽

Genotype Identification ◽

Host Selectivity ◽

Nad5 Gene ◽

Echinococcus Granulosus Sensu Lato

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

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Molecular Study of Echinococcus granulosus Cestodes in Ukraine and the First Genetic Identification of Echinococcus granulosus Sensu Stricto (G1 Genotype) in the Country

Acta Parasitologica ◽

10.1007/s11686-021-00450-z ◽

2021 ◽

Author(s):

Viliam Šnábel ◽

Tetiana A. Kuzmina ◽

Anatoliy A. Antipov ◽

Oleksandr M. Yemets ◽

Serena Cavallero ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Molecular Study ◽

Sensu Stricto ◽

Genetic Identification ◽

Echinococcus Granulosus Sensu Stricto

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Echinococcus granulosus sensu stricto: silencing of thioredoxin peroxidase impairs the differentiation of protoscoleces into metacestodes

Parasite ◽

10.1051/parasite/2018055 ◽

2018 ◽

Vol 25 ◽

pp. 57 ◽

Cited By ~ 5

Author(s):

Hui Wang ◽

Jun Li ◽

Chuanshan Zhang ◽

Baoping Guo ◽

Qin Wei ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Sensu Stricto ◽

Antioxidant Defence ◽

Culture Studies ◽

Antioxidant Defence System ◽

Thioredoxin Peroxidase ◽

Survival And Growth ◽

Echinococcus Granulosus Sensu Lato

Cystic echinococcosis (CE) is a cosmopolitan parasitic disease caused by infection with the larval stage of Echinococcus granulosus sensu lato. Thioredoxin peroxidase (TPx) may play an essential role in the antioxidant defence system of E. granulosus s.l. as neither catalase nor glutathione peroxidase activities have been detected in the parasite. However, it is not known whether TPx affects the survival and growth of E. granulosus s.l. during development. In this study, three fragments of siRNA specific for EgTPx (siRNA-1/2/3) were designed and transfected into protoscoleces of E. granulosus sensu stricto by electroporation. Quantitative real-time PCR and Western blotting analysis showed that siRNA-3 significantly reduced the expression of EgTPx. Coincidentally, knockdown of EgTPx expression in protoscoleces with siRNA-3 significantly reduced the viability of the parasite under oxidative stress induced by 0.6 mM H2O2. In vitro culture studies showed that protoscoleces treated with siRNA-3 reduced pre-microcyst formation. In vivo experiments showed that injecting mice intraperitoneally with protoscoleces treated with siRNA-3 resulted in a significant reduction in the number, size and weight of CE cysts compared with those of control animals. Silencing of EgTPx led to the impairment of growth of E. granulosus s.s. both in vitro and in vivo, indicating that EgTPx is an important factor for protoscoleces survival and plays an important role in the antioxidant defence against the host during development.

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The optimum cut-off value to differentiate Echinococcus granulosus sensu stricto from other species of E. granulosus sensu lato using larval rostellar hook morphometry

Journal of Helminthology ◽

10.1017/s0022149x13000473 ◽

2013 ◽

Vol 89 (1) ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

S.V. Soriano ◽

N.B. Pierangeli ◽

L.A. Pianciola ◽

M. Mazzeo ◽

L.E. Lazzarini ◽

...

Keyword(s):

Molecular Epidemiology ◽

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Control Strategies ◽

High Sensitivity ◽

Field Studies ◽

Sensu Stricto ◽

Roc Curve Analysis ◽

First Time

AbstractCystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1–G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6–G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 μm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

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