Cargando…

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein

BACKGROUND: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozo...

Descripción completa

Detalles Bibliográficos
Autores principales: Shrestha, Aruna, Palmieri, Nicola, Abd-Elfattah, Ahmed, Ruttkowski, Bärbel, Pagès, Marc, Joachim, Anja
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297187/
https://www.ncbi.nlm.nih.gov/pubmed/28173829
http://dx.doi.org/10.1186/s13071-017-2003-1
_version_ 1782505695109709824
author Shrestha, Aruna
Palmieri, Nicola
Abd-Elfattah, Ahmed
Ruttkowski, Bärbel
Pagès, Marc
Joachim, Anja
author_facet Shrestha, Aruna
Palmieri, Nicola
Abd-Elfattah, Ahmed
Ruttkowski, Bärbel
Pagès, Marc
Joachim, Anja
author_sort Shrestha, Aruna
collection PubMed
description BACKGROUND: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico. METHODS: The 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively. RESULTS: The CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite. CONCLUSIONS: The results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-017-2003-1) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5297187
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-52971872017-02-10 Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein Shrestha, Aruna Palmieri, Nicola Abd-Elfattah, Ahmed Ruttkowski, Bärbel Pagès, Marc Joachim, Anja Parasit Vectors Research BACKGROUND: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico. METHODS: The 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively. RESULTS: The CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite. CONCLUSIONS: The results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-017-2003-1) contains supplementary material, which is available to authorized users. BioMed Central 2017-02-07 /pmc/articles/PMC5297187/ /pubmed/28173829 http://dx.doi.org/10.1186/s13071-017-2003-1 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Shrestha, Aruna
Palmieri, Nicola
Abd-Elfattah, Ahmed
Ruttkowski, Bärbel
Pagès, Marc
Joachim, Anja
Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein
title Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein
title_full Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein
title_fullStr Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein
title_full_unstemmed Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein
title_short Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein
title_sort cloning, expression and molecular characterization of a cystoisospora suis specific uncharacterized merozoite protein
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297187/
https://www.ncbi.nlm.nih.gov/pubmed/28173829
http://dx.doi.org/10.1186/s13071-017-2003-1
work_keys_str_mv AT shresthaaruna cloningexpressionandmolecularcharacterizationofacystoisosporasuisspecificuncharacterizedmerozoiteprotein
AT palmierinicola cloningexpressionandmolecularcharacterizationofacystoisosporasuisspecificuncharacterizedmerozoiteprotein
AT abdelfattahahmed cloningexpressionandmolecularcharacterizationofacystoisosporasuisspecificuncharacterizedmerozoiteprotein
AT ruttkowskibarbel cloningexpressionandmolecularcharacterizationofacystoisosporasuisspecificuncharacterizedmerozoiteprotein
AT pagesmarc cloningexpressionandmolecularcharacterizationofacystoisosporasuisspecificuncharacterizedmerozoiteprotein
AT joachimanja cloningexpressionandmolecularcharacterizationofacystoisosporasuisspecificuncharacterizedmerozoiteprotein