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Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs

BACKGROUND: Human babesiosis is an infectious disease that is epidemic in various regions all over the world. The predominant causative pathogen of this disease is the intra-erythrocytic parasite Babesia microti. The thioredoxin system is one of the major weapons that is used in the resistance to th...

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Autores principales: Huang, Jingwei, Xiong, Kang, Zhang, Houshuang, Zhao, Yanzhen, Cao, Jie, Gong, Haiyan, Zhou, Yongzhi, Zhou, Jinlin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769273/
https://www.ncbi.nlm.nih.gov/pubmed/29335000
http://dx.doi.org/10.1186/s13071-018-2619-9
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author Huang, Jingwei
Xiong, Kang
Zhang, Houshuang
Zhao, Yanzhen
Cao, Jie
Gong, Haiyan
Zhou, Yongzhi
Zhou, Jinlin
author_facet Huang, Jingwei
Xiong, Kang
Zhang, Houshuang
Zhao, Yanzhen
Cao, Jie
Gong, Haiyan
Zhou, Yongzhi
Zhou, Jinlin
author_sort Huang, Jingwei
collection PubMed
description BACKGROUND: Human babesiosis is an infectious disease that is epidemic in various regions all over the world. The predominant causative pathogen of this disease is the intra-erythrocytic parasite Babesia microti. The thioredoxin system is one of the major weapons that is used in the resistance to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host immune system. In other intra-erythrocytic apicomplexans like the malaria parasite Plasmodium falciparum, anti-oxidative proteins are promising targets for the development of anti-parasitic drugs. However, to date, the sequences and biological properties of thioredoxins and thioredoxin-like molecules of B. microti remain unknown. Understanding the molecular characterization and function of B. microti thioredoxins may help to develop anti-Babesia drugs and controlling babesiosis. METHODS: The full-length B. microti thioredoxin 2 (BmTrx2) gene was obtained using a rapid amplification of cDNA ends (RACE) method, and the deduced BmTrx2 amino acid sequence was analyzed using regular bioinformatics tools. Recombinant BmTrx2 protein was expressed in vitro and purified using His-tag protein affinity chromatography resins. Reverse transcription PCR, quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx2. Indirect immunofluorescence assay was used to localize BmTrx2 in B. microti. Bovine insulin reduction assays were used to determine the enzyme activity of the purified recombinant BmTrx2 protein. RESULTS: The full-length BmTrx2 was 564 bp with a 408 bp open reading frame encoding a protein of 135 amino acids. The predicted molecular weight of the protein was 15.5 kDa. A conserved thioredoxin-like family domain was found in BmTrx2. The expression of BmTrx2 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. The results of Western blot analysis showed the native BmTrx2 in parasite lysates could be detected by mouse anti-BmTrx2 serum and that the recombinant BmTrx2 protein could be recognized by serum of B. microti-infected mice. Immunofluorescence microscopy showed that BmTrx2 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the purified recombinant BmTrx2 protein possesses antioxidant enzyme activity. Dihydroartemisinin and quinine, known anti-malaria drugs, and clindamycin, a known anti-babesiosis drug, induced significantly higher upregulation of BmTrx2 mRNA. CONCLUSIONS: Our results indicate that BmTrx2 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13071-018-2619-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-57692732018-01-25 Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs Huang, Jingwei Xiong, Kang Zhang, Houshuang Zhao, Yanzhen Cao, Jie Gong, Haiyan Zhou, Yongzhi Zhou, Jinlin Parasit Vectors Research BACKGROUND: Human babesiosis is an infectious disease that is epidemic in various regions all over the world. The predominant causative pathogen of this disease is the intra-erythrocytic parasite Babesia microti. The thioredoxin system is one of the major weapons that is used in the resistance to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host immune system. In other intra-erythrocytic apicomplexans like the malaria parasite Plasmodium falciparum, anti-oxidative proteins are promising targets for the development of anti-parasitic drugs. However, to date, the sequences and biological properties of thioredoxins and thioredoxin-like molecules of B. microti remain unknown. Understanding the molecular characterization and function of B. microti thioredoxins may help to develop anti-Babesia drugs and controlling babesiosis. METHODS: The full-length B. microti thioredoxin 2 (BmTrx2) gene was obtained using a rapid amplification of cDNA ends (RACE) method, and the deduced BmTrx2 amino acid sequence was analyzed using regular bioinformatics tools. Recombinant BmTrx2 protein was expressed in vitro and purified using His-tag protein affinity chromatography resins. Reverse transcription PCR, quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx2. Indirect immunofluorescence assay was used to localize BmTrx2 in B. microti. Bovine insulin reduction assays were used to determine the enzyme activity of the purified recombinant BmTrx2 protein. RESULTS: The full-length BmTrx2 was 564 bp with a 408 bp open reading frame encoding a protein of 135 amino acids. The predicted molecular weight of the protein was 15.5 kDa. A conserved thioredoxin-like family domain was found in BmTrx2. The expression of BmTrx2 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. The results of Western blot analysis showed the native BmTrx2 in parasite lysates could be detected by mouse anti-BmTrx2 serum and that the recombinant BmTrx2 protein could be recognized by serum of B. microti-infected mice. Immunofluorescence microscopy showed that BmTrx2 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the purified recombinant BmTrx2 protein possesses antioxidant enzyme activity. Dihydroartemisinin and quinine, known anti-malaria drugs, and clindamycin, a known anti-babesiosis drug, induced significantly higher upregulation of BmTrx2 mRNA. CONCLUSIONS: Our results indicate that BmTrx2 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13071-018-2619-9) contains supplementary material, which is available to authorized users. BioMed Central 2018-01-15 /pmc/articles/PMC5769273/ /pubmed/29335000 http://dx.doi.org/10.1186/s13071-018-2619-9 Text en © The Author(s). 2018 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
Huang, Jingwei
Xiong, Kang
Zhang, Houshuang
Zhao, Yanzhen
Cao, Jie
Gong, Haiyan
Zhou, Yongzhi
Zhou, Jinlin
Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs
title Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs
title_full Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs
title_fullStr Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs
title_full_unstemmed Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs
title_short Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs
title_sort molecular characterization of babesia microti thioredoxin (bmtrx2) and its expression patterns induced by antiprotozoal drugs
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769273/
https://www.ncbi.nlm.nih.gov/pubmed/29335000
http://dx.doi.org/10.1186/s13071-018-2619-9
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