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.