Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping.
Background Infection with Trypanosoma cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disea...
Published in: | PLOS Neglected Tropical Diseases |
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Online Access: | https://doi.org/10.1371/journal.pntd.0006388 https://doaj.org/article/a2fbb09216b04cdfb28b047579497d03 |
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ftdoajarticles:oai:doaj.org/article:a2fbb09216b04cdfb28b047579497d03 2023-05-15T15:14:06+02:00 Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. Fernanda Cristina Costa Amanda Fortes Francisco Shiromani Jayawardhana Simone Guedes Calderano Michael D Lewis Francisco Olmo Tom Beneke Eva Gluenz Jack Sunter Samuel Dean John Morrison Kelly Martin Craig Taylor 2018-04-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0006388 https://doaj.org/article/a2fbb09216b04cdfb28b047579497d03 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0006388 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006388 https://doaj.org/article/a2fbb09216b04cdfb28b047579497d03 PLoS Neglected Tropical Diseases, Vol 12, Iss 4, p e0006388 (2018) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2018 ftdoajarticles https://doi.org/10.1371/journal.pntd.0006388 2023-03-19T01:28:36Z Background Infection with Trypanosoma cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of T. cruzi biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite. Methodology/principal findings Here, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a T. cruzi reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the in vivo phenotype can be rapidly assessed. Conclusions/significance The techniques described here will have multiple applications for studying aspects of T. cruzi biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 12 4 e0006388 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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language |
English |
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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 Fernanda Cristina Costa Amanda Fortes Francisco Shiromani Jayawardhana Simone Guedes Calderano Michael D Lewis Francisco Olmo Tom Beneke Eva Gluenz Jack Sunter Samuel Dean John Morrison Kelly Martin Craig Taylor Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Background Infection with Trypanosoma cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of T. cruzi biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite. Methodology/principal findings Here, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a T. cruzi reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the in vivo phenotype can be rapidly assessed. Conclusions/significance The techniques described here will have multiple applications for studying aspects of T. cruzi biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a ... |
format |
Article in Journal/Newspaper |
author |
Fernanda Cristina Costa Amanda Fortes Francisco Shiromani Jayawardhana Simone Guedes Calderano Michael D Lewis Francisco Olmo Tom Beneke Eva Gluenz Jack Sunter Samuel Dean John Morrison Kelly Martin Craig Taylor |
author_facet |
Fernanda Cristina Costa Amanda Fortes Francisco Shiromani Jayawardhana Simone Guedes Calderano Michael D Lewis Francisco Olmo Tom Beneke Eva Gluenz Jack Sunter Samuel Dean John Morrison Kelly Martin Craig Taylor |
author_sort |
Fernanda Cristina Costa |
title |
Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. |
title_short |
Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. |
title_full |
Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. |
title_fullStr |
Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. |
title_full_unstemmed |
Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. |
title_sort |
expanding the toolbox for trypanosoma cruzi: a parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined crispr/cas9 functionality for rapid in vivo localisation and phenotyping. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2018 |
url |
https://doi.org/10.1371/journal.pntd.0006388 https://doaj.org/article/a2fbb09216b04cdfb28b047579497d03 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 12, Iss 4, p e0006388 (2018) |
op_relation |
https://doi.org/10.1371/journal.pntd.0006388 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006388 https://doaj.org/article/a2fbb09216b04cdfb28b047579497d03 |
op_doi |
https://doi.org/10.1371/journal.pntd.0006388 |
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PLOS Neglected Tropical Diseases |
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12 |
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4 |
container_start_page |
e0006388 |
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