Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping.
Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be e...
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ftdoajarticles:oai:doaj.org/article:789058e0d4f341bd824d5e232f02ac65 2023-05-15T15:12:27+02:00 Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. Helder Magno Silva Valadares Juliana Ramos Pimenta Marcela Segatto Vanja Maria Veloso Mônica Lúcia Gomes Egler Chiari Kenneth John Gollob Maria Terezinha Bahia Marta de Lana Glória Regina Franco Carlos Renato Machado Sérgio Danilo Junho Pena Andréa Mara Macedo 2012-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0001722 https://doaj.org/article/789058e0d4f341bd824d5e232f02ac65 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC3393670?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0001722 https://doaj.org/article/789058e0d4f341bd824d5e232f02ac65 PLoS Neglected Tropical Diseases, Vol 6, Iss 7, p e1722 (2012) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2012 ftdoajarticles https://doi.org/10.1371/journal.pntd.0001722 2022-12-31T14:07:43Z Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations--Silvio X10 cl1 (TcI) and Esmeraldo cl3 (TcII)--and three naturally occurring strains, one isolated from a vector (A316A R7) and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 6 7 e1722 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Helder Magno Silva Valadares Juliana Ramos Pimenta Marcela Segatto Vanja Maria Veloso Mônica Lúcia Gomes Egler Chiari Kenneth John Gollob Maria Terezinha Bahia Marta de Lana Glória Regina Franco Carlos Renato Machado Sérgio Danilo Junho Pena Andréa Mara Macedo Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations--Silvio X10 cl1 (TcI) and Esmeraldo cl3 (TcII)--and three naturally occurring strains, one isolated from a vector (A316A R7) and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality. |
format |
Article in Journal/Newspaper |
author |
Helder Magno Silva Valadares Juliana Ramos Pimenta Marcela Segatto Vanja Maria Veloso Mônica Lúcia Gomes Egler Chiari Kenneth John Gollob Maria Terezinha Bahia Marta de Lana Glória Regina Franco Carlos Renato Machado Sérgio Danilo Junho Pena Andréa Mara Macedo |
author_facet |
Helder Magno Silva Valadares Juliana Ramos Pimenta Marcela Segatto Vanja Maria Veloso Mônica Lúcia Gomes Egler Chiari Kenneth John Gollob Maria Terezinha Bahia Marta de Lana Glória Regina Franco Carlos Renato Machado Sérgio Danilo Junho Pena Andréa Mara Macedo |
author_sort |
Helder Magno Silva Valadares |
title |
Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. |
title_short |
Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. |
title_full |
Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. |
title_fullStr |
Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. |
title_full_unstemmed |
Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping. |
title_sort |
unequivocal identification of subpopulations in putative multiclonal trypanosoma cruzi strains by facs single cell sorting and genotyping. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2012 |
url |
https://doi.org/10.1371/journal.pntd.0001722 https://doaj.org/article/789058e0d4f341bd824d5e232f02ac65 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 6, Iss 7, p e1722 (2012) |
op_relation |
http://europepmc.org/articles/PMC3393670?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0001722 https://doaj.org/article/789058e0d4f341bd824d5e232f02ac65 |
op_doi |
https://doi.org/10.1371/journal.pntd.0001722 |
container_title |
PLoS Neglected Tropical Diseases |
container_volume |
6 |
container_issue |
7 |
container_start_page |
e1722 |
_version_ |
1766343130487980032 |