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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

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Autor(es):
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Costa, Fernanda Cristina [1, 2] ; Francisco, Amanda Fortes [1] ; Jayawardhana, Shiromani [1] ; Calderano, Simone Guedes [3, 1] ; Lewis, Michael D. [1] ; Olmo, Francisco [1] ; Beneke, Tom [4] ; Gluenz, Eva [4] ; Sunter, Jack [5] ; Dean, Samuel [4] ; Kelly, John Morrison [1] ; Taylor, Martin Craig [1]
Número total de Autores: 12
Afiliação do(s) autor(es):
[1] London Sch Hyg & Trop Med, Fac Infect & Trop Dis, London - England
[2] Univ Sao Paulo, Inst Phys Sao Carlos, Sao Carlos - Brazil
[3] Inst Butantan, Parasitol Lab, Sao Paulo - Brazil
[4] Univ Oxford, Sir William Dunn Sch Pathol, Oxford - England
[5] Oxford Brookes Univ, Dept Biol & Med Sci, Oxford - England
Número total de Afiliações: 5
Tipo de documento: Artigo Científico
Fonte: PLoS Neglected Tropical Diseases; v. 12, n. 4 APR 2018.
Citações Web of Science: 10
Resumo

Background Infection with Trypanosome 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 community resource and are freely available on request. (AU)

Processo FAPESP: 16/21283-9 - Desenvolvimento de linhagem de T. cruzi com expressão induzível de Cas9: uma ferramenta para edição genética
Beneficiário:Simone Guedes Calderano
Linha de fomento: Bolsas no Exterior - Pesquisa
Processo FAPESP: 16/08958-7 - Explorando o uso de nova tecnologia de edição de genoma como uma abordagem rápida para o estudo da biologia de topoisomerases em Trypanosoma Cruzi
Beneficiário:Fernanda Cristina Costa
Linha de fomento: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado
Processo FAPESP: 13/07600-3 - CIBFar - Centro de Inovação em Biodiversidade e Fármacos
Beneficiário:Glaucius Oliva
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs