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Biochemical characterization and differential expression of a 16.5-kilodalton tegument-associated antigen from the liver fluke Fasciola hepatica.

ABSTRACT: A cDNA encoding a 16.5-kDa protein termed FhTP16.5 was identified by immunoscreening of a cDNA library from Fasciola hepatica adult flukes using pooled sera from rabbits infected with F. hepatica for 4 weeks. Quantitative reverse transcriptase PCR (qPCR) analysis revealed that FhTP16.5 is not expressed in unembryonated eggs. It is poorly expressed in miracidia and highly expressed at the juvenile and adult stages; however, significant differences were found between the expression levels of FhTP16.5 in juveniles versus adult flukes. Recombinant FhTP16.5 was expressed at high levels in Escherichia coli, purified by affinity chromatography, and used to raise anti-FhTP16.5 polyclonal antibodies in rabbits. Immunoblot analysis using the anti-FhTP16.5 IgG antibody identified FhTP16.5 in crude and tegumental extracts and in excretory-secretory products of F. hepatica. The protein was not detected in crude extracts of Schistosoma mansoni or Schistosoma japonicum. Antibodies to FhTP16.5 were detected in the sera of rabbits at 3 to 12 weeks of F. hepatica infection as well as in the sera of humans with chronic fascioliasis; these findings suggest that FhTP16.5 could be a good antigen for serodiagnosis of fascioliasis. Immunohistochemistry demonstrated that FhTP16.5 localizes to the surface of the tegument of various developmental stages and in parenchymal tissues of the adult fluke. Such specific localization makes FhTP16.5 an attractive target for immunoprophylaxis or chemotherapy.

SUBMITTER: Gaudier JF

PROVIDER: S-EPMC3294618 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Biochemical characterization and differential expression of a 16.5-kilodalton tegument-associated antigen from the liver fluke Fasciola hepatica.

Gaudier José F JF Cabán-Hernández Kimberly K Osuna Antonio A Espino Ana M AM

Clinical and vaccine immunology : CVI 20120125 3

A cDNA encoding a 16.5-kDa protein termed FhTP16.5 was identified by immunoscreening of a cDNA library from Fasciola hepatica adult flukes using pooled sera from rabbits infected with F. hepatica for 4 weeks. Quantitative reverse transcriptase PCR (qPCR) analysis revealed that FhTP16.5 is not expressed in unembryonated eggs. It is poorly expressed in miracidia and highly expressed at the juvenile and adult stages; however, significant differences were found between the expression levels of FhTP1 ...[more]

PMID: 22278327

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Project description:BACKGROUND:Snails such as Galba truncatula are hosts for trematode flukes causing fascioliasis, a zoonosis that is a major public health problem. Galba truncatula has recently been shown to be a cryptic species complex. African populations of Galba spp. are not yet studied using molecular assessments and is imperative to do so and reconstruct the centre of origin of Galba and to understand when and by what means it may have colonized the highlands of Africa and to what extent humans might have been involved in that process. METHODS:Samples from all known sub-ranges throughout Africa and new samples from Europe and Asia were obtained. We used a combination of two mitochondrial (cox1 and 16S) and one nuclear (ITS2) markers and phylogenetic, divergence time estimates and phylogeographical methods to determine the identity and biogeographical affinities. We also reconstructed the colonization history including the likely mode of dispersal and tested for the presence of cryptic Galba species in Africa. RESULTS:Galba truncatula is restricted to the Palaearctic region of the continent, namely Morocco. All sub-Saharan populations proved to be a distinct species according to the phylogenetic analyses and genetic distance. We propose to use the existing name Galba mweruensis (Connolly, 1929) for this species which is morphologically indistinguishable from the other two species hitherto known to occur in northern Africa, i.e. G. truncatula and G. schirazensis. Sub-tropical Africa has been colonized only once in either the Pliocene and possibly Miocene. Diversification within G. mweruensis is dated to the Plio-Pleistocene and thus human-mediated dispersal can be ruled out for the initial colonization of the isolated mountain ranges. There are potentially even more cryptic species in high altitude areas of Africa as outlined by the distinctness of the population found at the top of Mt. Elgon, Uganda. CONCLUSIONS:From a novel genetic inspection of available African material, a hitherto neglected distinct species, G. mweruensis, now appears a major host of F. hepatica throughout sub-Saharan Africa. A closer examination of trematode parasites hosted by this species is needed in order to understand transmission patterns in highlands throughout eastern and southern Africa. We encourage future studies to inspect other high altitudes areas in Africa in light of parasites of either veterinary or medical importance.

Project description:BackgroundThe common liver fluke Fasciola hepatica is the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism. Genomics and transcriptomics are still in their infancy in F. hepatica, with very scarce information available from the invasive newly excysted juveniles (NEJ). Here we provide an initial glimpse to the transcriptomics of the NEJ, the first stage to interact with the mammalian host.ResultsWe catalogued more than 500 clusters generated from the analysis of F. hepatica juvenile expressed sequence tags (EST), several of them not detected in the adult stage. A set of putative F. hepatica specific transcripts, and a group of sequences conserved exclusively in flatworms were identified. These novel sequences along with a set of parasite transcripts absent in the host genomes are putative new targets for future anti-parasitic drugs or vaccine development. Comparisons of the F. hepatica sequences with other metazoans genomes or EST databases were consistent with the basal positioning of flatworms in the bilaterian phylogeny. Notably, GC content, codon usage and amino acid frequencies are remarkably different in Schistosomes to F. hepatica and other trematodes. Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signaling and mitochondrial enzymes were identified. Differential expression of secreted protease gene family members between juvenile and adult stages may respond to different needs during host colonization.ConclusionThe knowledge of the genes expressed by the invasive stage of Fasciola hepatica is a starting point to unravel key aspects of this parasite's biology. The integration of the emerging transcriptomics, and proteomics data and the advent of functional genomics tools in this organism are positioning F. hepatica as an interesting model for trematode biology.

Dataset Information

Biochemical characterization and differential expression of a 16.5-kilodalton tegument-associated antigen from the liver fluke Fasciola hepatica.

Publications

Biochemical characterization and differential expression of a 16.5-kilodalton tegument-associated antigen from the liver fluke Fasciola hepatica.

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