Discussion

Studies on the pathogenesis and immunology of chronic murine Chagas disease are limited by the scarcity of parasites which complicates analysis of local responses to infected cells. We recently developed highly sensitive bioluminescent (BLI) in vivo imaging models for Trypanosoma cruzi using a red-shifted luciferase variant (PpyRE9h). However, the utility of such models is limited by the low resolution of BLI at the level of individual infected host cells. To circumvent this problem, we have generated T. cruzi expressing a fusion protein consisting of an N-terminal luciferase and a C-terminal green fluorescent domain. The bioluminescence of the new parasite line is unchanged by the fusion, the green fluorescent domain has no effect on the wavelength of bioluminescent emission, and the fusion protein is expressed throughout the life cycle. The modified parasites show identical infection characteristics to the original bioluminescent line. The dual reporter system allows the sites of tissue-specific infection in mice to be visualised (based on bioluminescence), and the identification and isolation of infected cells (based on fluorescence). Intriguingly, approximately 50% of infected cells contained an uneven number of amastigotes suggesting that intracellular parasite replication is asynchronous. Extracellular amastigote-like forms with a short protruding flagellum and epimastigote&hypen;like forms could also be detected in infected mice. We are currently developing protocols to optimise the isolation and phenotyping of infected host cells. These techniques will provide new approaches for investigating the mechanisms of immune evasion and parasite persistence during the chronic phase of the disease.