Dataset Information

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti.

ABSTRACT: A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

SUBMITTER: Doleckova K

PROVIDER: S-EPMC2625449 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti.

Dolecková Katerina K Kasný Martin M Mikes Libor L Cartwright Jared J Jedelský Petr P Schneider Eric L EL Dvorák Jan J Mountford Adrian P AP Craik Charles S CS Horák Petr P

International journal for parasitology 20080726 2

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluoro ...[more]

PMID: 18708063

Similar Datasets

Project description:BackgroundCercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen Schistosoma mansoni. As bird schistosomes can also penetrate human skin and cause cercarial dermatitis, our aim was to characterize the architecture and ultrastructure of glands in the neurotropic bird schistosome Trichobilharzia regenti and compare it with S. mansoni. In the context of different histolytic enzymes used by these two species, we focused also on the estimations of gland volumes and pH in T. regenti.ResultsThe architecture and 3-D models of two types of acetabular penetration glands, their ducts and of the head gland are shown here. We characterized secretory vesicles in all three gland types by means of TEM and confirmed accuracy of the models obtained by confocal microscopy. The results of two independent approaches showed that the glands occupy ca. one third of cercarial body volume (postacetabular glands ca. 15%, circumacetabular 12% and head gland 6%). The inner environment within the two types of acetabular glands differed significantly as evidenced by dissimilar ability to bind fluorescent markers and by pH value which was higher in circumacetabular (7.44) than in postacetabular (7.08) glands.ConclusionsAs far as we know, this is the first presentation of a 3-D model of cercarial glands and the first exact estimation of the volumes of the three gland types in schistosomes. Our comparisons between T. regenti and S. mansoni implied that the architecture and ultrastructure of the glands is most likely conserved within the family. Only minor variations were found between the two species. It seems that the differences in molecular composition have no effect on general appearance of the secretory cells in TEM. Fluorescent markers employed in this study, distinguishing between secretory vesicles and gland types, can be useful in further studies of mechanisms used by cercariae for host invasion. Results of the first attempts to estimate pH within schistosome glands may help further understanding of regulation of enzymatic activities present within the glands.

Project description:Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools.

Project description:The invasive larvae (cercariae) of schistosomes penetrate the skin of their definitive hosts. During the invasion, they undergo dramatic ultrastructural and physiological transitions. These changes result in the development of the subsequent stage, schistosomulum, which migrates through host tissues in close contact with host's immune system. One of the striking changes in the transforming cercariae is the shedding of their thick tegumental glycocalyx, which represents an immunoattractive structure; therefore its removal helps cercariae to avoid immune attack. A set of commercial fluorescently labeled lectin probes, their saccharide inhibitors and monoclonal antibodies against the trisaccharide Lewis-X antigen (LeX, CD15) were used to characterize changes in the surface saccharide composition of the neuropathogenic avian schistosome Trichobilharzia regenti during the transformation of cercariae to schistosomula, both in vitro and in vivo. The effect of various lectins on glycocalyx shedding was evaluated microscopically. The involvement of peptidases and their inhibitors on the shedding of glycocalyx was investigated using T. regenti recombinant cathepsin B2 and a set of peptidase inhibitors. The surface glycocalyx of T. regenti cercariae was rich in fucose and mannose/glucose residues. After the transformation of cercariae in vitro or in vivo within their specific duck host, reduction and vanishing of these epitopes was observed, and galactose/N-acetylgalactosamine emerged. The presence of LeX was not observed on the cercariae, but the antigen was gradually expressed from the anterior part of the body in the developing schistosomula. Some lectins which bind to the cercarial surface also induced secretion from the acetabular penetration glands. Seven lectins induced the shedding of glycocalyx by cercariae, among which five bound strongly to cercarial surface; the effect could be blocked by saccharide inhibitors. Mannose-binding protein, part of the lectin pathway of the complement system, also bound to cercariae and schistosomula, but had little effect on glycocalyx shedding. Our study did not confirm the involvement of proteolysis in glycocalyx shedding.

Dataset Information

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti.

Publications

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets