Excretory–secretory products of larval Fasciola hepatica investigated using a two-dimensional proteomic approach

ABSTRACT The liver fluke Fasciola hepatica parasitizes humans and ruminant livestock worldwide, and it is now being considered a reemerging zoonotic disease, especially in areas in which it is endemic, such as South America. This study investigates the immune response to excretory and secretory products produced by F. hepatica in a group of patients from the Peruvian Altiplano, where the disease is highly endemic. Using a proteomic approach and immunoblotting techniques, we have identified the enzymes leucine aminopeptidase (LAP) and phosphoenolpyruvate carboxykinase as immunodominant antigens recognized by sera from fasciolosis patients. An indirect enzyme-linked immunosorbent assay using recombinant LAP as the antigen was developed to check sera from individuals of this region. Our results demonstrate that LAP produces a specific and strong reaction, suggesting its potential use in the serologic diagnosis of F. hepatica infections in humans.

Download Full-text

Protein Detection of Excretory-Secretory Products and Somatic Extracts from Fasciola hepatica and F. gigantica Using Two-Dimensional Electrophoresis

Iranian Journal of Parasitology ◽

10.18502/ijpa.v14i3.1476 ◽

2019 ◽

Author(s):

Afshin RASOULI ◽

Ali FARAHNAK ◽

Hakimeh ZALI ◽

Mostafa REZAEIAN ◽

Abolfazl GOLESTANI ◽

...

Keyword(s):

Vaccine Development ◽

Fasciola Hepatica ◽

Culture Media ◽

Protein Detection ◽

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Protease Enzyme ◽

Secretory Products ◽

Dimensional Electrophoresis ◽

Somatic Extract

Background: The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. Methods: Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017-2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. Results: For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. Conclusion: The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development.

Download Full-text

Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics

Pathogens ◽

10.3390/pathogens10060725 ◽

2021 ◽

Vol 10 (6) ◽

pp. 725

Author(s):

David Becerro-Recio ◽

Javier González-Miguel ◽

Alberto Ucero ◽

Javier Sotillo ◽

Álvaro Martínez-Moreno ◽

...

Keyword(s):

Experimental Infection ◽

Fasciola Hepatica ◽

Cathepsin L ◽

Helminth Parasites ◽

Glutathione S Transferase ◽

Serum Samples ◽

Secretory Products ◽

Immunogenic Proteins ◽

Host Parasite ◽

First Time

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

Download Full-text

O-GlcNAcylation confers protection againstStaphylococcus aureusinfection inCaenorhabditis elegansthrough ubiquitination

RSC Advances ◽

10.1039/c8ra00279g ◽

2018 ◽

Vol 8 (41) ◽

pp. 23089-23100 ◽

Cited By ~ 6

Author(s):

Loganathan Vigneshwari ◽

Boopathi Balasubramaniam ◽

Sivasamy Sethupathy ◽

Shunmugiah Karutha Pandian ◽

Krishnaswamy Balamurugan

Keyword(s):

Staphylococcus Aureus ◽

Caenorhabditis Elegans ◽

Two Dimensional ◽

Proteomic Approach ◽

Ubiquitination Pathway

Two-dimensional gel-based proteomic approach unveiled that,O-GlcNAcylation protectsCaenorhabditis elegansfromStaphylococcus aureusinfection by upregulating the proteins involved in ubiquitination pathway.

Download Full-text

Partial Characterization of the Epitope on Excretory-Secretory Products of Fasciola hepatica Recognized by Monoclonal Antibody ES78

Journal of Parasitology ◽

10.2307/3284530 ◽

1998 ◽

Vol 84 (1) ◽

pp. 55 ◽

Cited By ~ 17

Author(s):

Ailen Diaz ◽

Ana M. Espino ◽

Ricardo Marcet ◽

Oscar Otero ◽

Diamela Torres ◽

...

Keyword(s):

Monoclonal Antibody ◽

Fasciola Hepatica ◽

Partial Characterization ◽

Secretory Products

Download Full-text

Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs

Reproduction Fertility and Development ◽

10.1071/rd03079 ◽

2004 ◽

Vol 16 (2) ◽

pp. 69 ◽

Cited By ~ 7

Author(s):

S. A. Coonrod ◽

M. E. Calvert ◽

P. P. Reddi ◽

E. N. Kasper ◽

L. C. Digilio ◽

...

Keyword(s):

Plasma Membrane ◽

Indirect Immunofluorescence ◽

Zona Pellucida ◽

Surface Expression ◽

Surface Proteins ◽

2D Electrophoresis ◽

Dependent Manner ◽

Two Dimensional ◽

Phase Partitioning ◽

Proteomic Approach

In order to gain a deeper understanding of the molecular underpinnings of sperm–egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode’s solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning. Immunoprecipitation assays also demonstrated that surface re-mZP3 was released from transfected CV-1 in a time-dependent manner. These results demonstrate that ZP3 is specifically associated with the surface of mature eggs and its subsequent release from the cell surface may represent one mechanism by which ZP3 is secreted. Furthermore, the increase in ZP3 surface expression following fertilisation suggests that ZP3 may have a functional role during sperm–oolemma binding and fusion. These results also validate the usefulness of using the 2D proteomic approach to identify and characterise egg-surface proteins.

Download Full-text