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

Costa, F. C. et al. (2018) 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. PLoS Neglected Tropical Diseases, 12(4), e0006388. (doi: 10.1371/journal.pntd.0006388) (PMID:29608569) (PMCID:PMC5897030)

Abstract

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 community resource and are freely available on request.

• Item Type: Articles
• Additional Information: Funding: This work was funded by British Heart Foundation grant no. PG/13/88/30556 to JMK (www.bhf.org.uk/). FCC and SGC were funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (CEPID grant 2013/07600-3 and fellowship 2016/08958-7 to FCC and 2016/21283-9 to SGC (www.fapesp.br/)). FO was funded by the Fundación Alonso Martin Escudero (http://www.fundame.org). MDL was funded by a European Union FP7 Marie-Curie Actions award 625810 (http://ec.europa.eu/research/mariecurieactions/). EG is a Royal Society University Research Fellow (https://royalsociety.org/) and TB is supported by a MRC PhD studentships (15/16_MSD_836338, www.mrc.ac.uk). JS was funded by an MRC Newton Fund award [MR/N017323/1, www.newtonfund.ac.uk/]. JS and SD were funded by the Wellcome Trust grant nos. 108445/Z/15/Z (awarded to SD, JS and Professor Keith Gull, and 104627/Z/14/Z awarded to Professor Keith Gull https://wellcome.ac.uk/).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Gluenz, Dr Eva

Creator Roles

Gluenz, E.: Methodology, Resources, Writing – review and editing

• Authors: Costa, F. C., Francisco, A. F., Jayawardhana, S., Calderano, S. G., Lewis, M. D., Olmo, F., Beneke, T., Gluenz, E., Sunter, J., Dean, S., Kelly, J. M., and Taylor, M. C.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: PLoS Neglected Tropical Diseases
• Publisher: Public Library of Science
• ISSN: 1935-2727
• ISSN (Online): 1935-2735
• First Published: First published in PLoS Neglected Tropical Diseases
• Publisher Policy: Reproduced under a Creative Commons license