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Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient
BACKGROUND: Plasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosq...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249376/ https://www.ncbi.nlm.nih.gov/pubmed/32450861 http://dx.doi.org/10.1186/s12936-020-03237-1 |
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author | Zhou, Yaxian Grieser, Alexis M. Do, Julie Itsara, Leslie S. Vaughan, Ashley M. Ghosh, Anil K. |
author_facet | Zhou, Yaxian Grieser, Alexis M. Do, Julie Itsara, Leslie S. Vaughan, Ashley M. Ghosh, Anil K. |
author_sort | Zhou, Yaxian |
collection | PubMed |
description | BACKGROUND: Plasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates. METHODS: Plasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28. RESULTS: After stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28. CONCLUSION: The combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium. |
format | Online Article Text |
id | pubmed-7249376 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-72493762020-06-04 Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient Zhou, Yaxian Grieser, Alexis M. Do, Julie Itsara, Leslie S. Vaughan, Ashley M. Ghosh, Anil K. Malar J Methodology BACKGROUND: Plasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates. METHODS: Plasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28. RESULTS: After stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28. CONCLUSION: The combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium. BioMed Central 2020-05-25 /pmc/articles/PMC7249376/ /pubmed/32450861 http://dx.doi.org/10.1186/s12936-020-03237-1 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. |
spellingShingle | Methodology Zhou, Yaxian Grieser, Alexis M. Do, Julie Itsara, Leslie S. Vaughan, Ashley M. Ghosh, Anil K. Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient |
title | Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient |
title_full | Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient |
title_fullStr | Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient |
title_full_unstemmed | Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient |
title_short | Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient |
title_sort | purification and production of plasmodium falciparum zygotes from in vitro culture using magnetic column and percoll density gradient |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249376/ https://www.ncbi.nlm.nih.gov/pubmed/32450861 http://dx.doi.org/10.1186/s12936-020-03237-1 |
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