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Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus

BACKGROUND: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas dis...

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Autores principales: Moreira, Carlos JC, Waniek, Peter J, Valente, Richard H, Carvalho, Paulo C, Perales, Jonas, Feder, Denise, Geraldo, Reinaldo B, Castro, Helena C, Azambuja, Patricia, Ratcliffe, Norman A, Mello, Cícero B
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898217/
https://www.ncbi.nlm.nih.gov/pubmed/24423259
http://dx.doi.org/10.1186/1756-3305-7-23
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author Moreira, Carlos JC
Waniek, Peter J
Valente, Richard H
Carvalho, Paulo C
Perales, Jonas
Feder, Denise
Geraldo, Reinaldo B
Castro, Helena C
Azambuja, Patricia
Ratcliffe, Norman A
Mello, Cícero B
author_facet Moreira, Carlos JC
Waniek, Peter J
Valente, Richard H
Carvalho, Paulo C
Perales, Jonas
Feder, Denise
Geraldo, Reinaldo B
Castro, Helena C
Azambuja, Patricia
Ratcliffe, Norman A
Mello, Cícero B
author_sort Moreira, Carlos JC
collection PubMed
description BACKGROUND: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. METHODS: SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. RESULTS: The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1′ was translocated to the putative position P4′-P5′. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. CONCLUSIONS: For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.
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spelling pubmed-38982172014-01-23 Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus Moreira, Carlos JC Waniek, Peter J Valente, Richard H Carvalho, Paulo C Perales, Jonas Feder, Denise Geraldo, Reinaldo B Castro, Helena C Azambuja, Patricia Ratcliffe, Norman A Mello, Cícero B Parasit Vectors Research BACKGROUND: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. METHODS: SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. RESULTS: The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1′ was translocated to the putative position P4′-P5′. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. CONCLUSIONS: For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule. BioMed Central 2014-01-14 /pmc/articles/PMC3898217/ /pubmed/24423259 http://dx.doi.org/10.1186/1756-3305-7-23 Text en Copyright © 2014 Moreira et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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
Moreira, Carlos JC
Waniek, Peter J
Valente, Richard H
Carvalho, Paulo C
Perales, Jonas
Feder, Denise
Geraldo, Reinaldo B
Castro, Helena C
Azambuja, Patricia
Ratcliffe, Norman A
Mello, Cícero B
Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus
title Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus
title_full Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus
title_fullStr Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus
title_full_unstemmed Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus
title_short Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus
title_sort isolation and molecular characterization of a major hemolymph serpin from the triatomine, panstrongylus megistus
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898217/
https://www.ncbi.nlm.nih.gov/pubmed/24423259
http://dx.doi.org/10.1186/1756-3305-7-23
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