Proteomic study of activated Taenia solium oncospheres

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Abstract

Taenia solium cysticerci are a major cause of human seizures and epilepsy in the world. In the gastrointestinal tract of infected individuals, taeniid eggs release the oncospheres, which are then activated by intestinal stimuli, getting ready to penetrate the gut wall and reach distant locations where they transform in cysticerci. Information about oncospheral molecules is scarce, and elucidation of the oncosphere proteome could help understanding the host–parasite relationship during the first steps of infection. In this study, using liquid chromatography and tandem mass spectrometry (LC–MS/MS) analysis, we could identify a set of oncospheral proteins involved in adhesion, protein folding, detoxification and proteolysis, among others. In addition, we have characterized one of the identified molecules, the parasite 14-3-3, by immunoblot and immunolocalization. The identification of these oncospheral proteins represents the first step to elucidate their specific roles in the biology of the host–parasite relationship.

Graphical abstract

By proteome techniques we identify a set of Taenia solium oncospheral proteins and further characterize the parasite 14-3-3, providing new information about the host–parasite relationship.

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Introduction

Taenia solium and Taenia saginata are among the most prevalent human tapeworms in the world. The intermediate host of T. solium is mainly pigs. Humans act as definitive hosts for the parasite. Gravid segments from humans infected with the adult tapeworm are passed in the faeces each containing around 40,000 eggs that resist destruction in the environment for a relatively long period of time. After ingestion by an intermediate host, oncospheres hatch and activate in the intestine, penetrate the gut and reach different organs via the blood stream, developing into cysticerci mainly in striated muscle. Humans are infected when ingesting raw or inadequately cooked pork containing viable cysticerci. The humans may also act as intermediate hosts for the parasite due to the accidental ingestion of T. solium eggs via unwashed hands or contaminated food, giving rise to human cysticercosis. While infection of the intestinal tract by adult worms is not life-threatening, infection with larval tapeworms from T. solium in man can be very serious and can lead to death when these localize in brain tissue.

Oncospheral hatching and activation have been achieved in vitro, showing that activated oncospheres are surrounded by parasite-derived excretory vesicles [1]. Little is known about the molecular cross-talk between taeniid oncospheres and its host when parasites are activated and ready to traverse the intestinal tissue. In this regard, genomic and ESTs studies have been recently reported for adult worms and cysticerci [2], [3], but not for oncospheres. Similarly, no proteomic studies on T. solium have been performed to date, and thus only individual proteins have been identified and characterized, and from those, the majority have been investigated in cysticerci and adult worms. The cellular events occurring within seconds to minutes after oncospheral activation are probably similar to those found in other eukaryotes upon “activation”, and most probably include changes in intracellular physiology, signaling through kinases and secondary messengers, remodelling of the cytoskeleton and an increase in the overall metabolic rate of the organism [e.g. 4]. In addition, molecules present in the penetration glands of oncospheres, and also those related with adhesion, proteolysis, and sorting-folding of proteins in secreted granules, would be expected to play key roles in the mechanism of oncospheral gut invasion. Oncospheres are the parasite stage determining the infection success in their intermediate host, and thus the identification of proteins from activated oncospheres is the key to understand both the parasite biology and the host–parasite relationship. We initiate here the study of the proteome from in vitro activated oncospheres. To achieve this goal, in vitro hatched and activated oncospheres of T. solium were treated with trypsin and the peptides released were analysed by LC–MS/MS for their identification. In addition, we further characterize one of the parasite proteins – the 14-3-3 – found during our proteomic approach.

Section snippets

Parasites

T. solium adult worms fragments (gravid proglottids) were recovered from Peruvian patients’ stools as described previously [5]. The proglottids were collected by sieving, washed thoroughly with distilled water and stored in 25% glycerol supplemented with penicillin (1000 IU/ml) and gentamicin (100 μg/ml) at 4 °C until used. Species identification was made by histology and PCR-RFLP as described before [5], [6]. The eggs were obtained from gravid proglottids by gentle homogenization (using a manual

Appearance and viability of oncospheres after activation

Activated oncospheres (90% of hatched oncospheres) showed a round-shaped appearance, border-located hooks and the absence of the oncospheral membrane (physically disrupted and discarded, as expected), under a microscope at 100× (Fig. 1a). In addition, activated parasites occasionally showed the presence of secretory vesicles around them (Fig. 1b). Around 10% of oncospheres showed the outer envelope and were considered as hatched but non-activated parasites (data not shown). Activated

Discussion

In an attempt to give the first picture of the biology of invasion by T. solium oncospheres, we have performed a proteomic analysis from in vitro activated oncospheres. Our activation approach resulted in a parasite sample consisting of 90% activated oncospheres without oncospheral membrane, together with 10% activated but “unreleased” parasites presenting the oncospheral membrane. Although these were a minority, we cannot rule out that some of the identified proteins are present in the

Acknowledgements

This project was partially funded by the Fogarty International Center/NIH (training grants D43 TW001140, TW007490 and TW006581), and the Bill and Melinda Gates Foundation (training grant #33848). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the sponsors. AHG acknowledges financial support from the CSIC-Predoctorate JAE Spanish grant programme.

References (45)

  • M.W. Lightowlers

    Vaccines for prevention of cysticercosis

    Acta Trop

    (2003)
  • R.S. Curwen et al.

    The Schistosoma mansoni soluble proteome: a comparison across four life-cycle stages

    Mol Biochem Parasitol

    (2004)
  • R.S. Curwen et al.

    Identification of novel proteases and immunomodulators in the secretions of schistosome cercariae that facilitate host entry

    Mol Cell Proteomics

    (2006)
  • J. Takagi

    Structural basis for ligand recognition by integrins

    Curr Opin Cell Biol

    (2007)
  • E.E. Avila et al.

    Entamoeba histolytica trophozoites: a surface-associated cysteine protease

    Exp Parasitol

    (1993)
  • M.B. Maes et al.

    Dipeptidyl peptidase II (DPPII), a review

    Clin Chim Acta

    (2007)
  • V. Pancholi et al.

    Housekeeping enzymes as virulence factors for pathogens

    Int J Med Microbiol

    (2003)
  • M. Siles-Lucas et al.

    The 14-3-3 protein: a key molecule in parasites as in other organisms

    Trends Parasitol

    (2003)
  • M. Siles-Lucas et al.

    14-3-3 proteins in Echinococcus: their role and potential as protective antigens

    Exp Parasitol

    (2008)
  • M. Siles-Lucas et al.

    Stage-specific expression of the 14-3-3 gene in Echinococcus multilocularis

    Mol Biochem Parasitol

    (1998)
  • F. Liu et al.

    Excretory/secretory proteome of the adult developmental stage of human blood fluke, Schistosoma japonicum

    Mol Cell Proteomics

    (2009)
  • M. Verastegui et al.

    Taenia solium oncosphere adhesion to intestinal epithelial and Chinese hamster ovary cells in vitro

    Infect Immun

    (2007)
  • Cited by (0)

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