Exploiting Genetically Modified Dual-Reporter Strains to Monitor Experimental Trypanosoma cruzi Infections and Host-Parasite Interactions

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase that emits tissue-penetrating orange-red light. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. This technology has allowed chronic infections to be followed in real-time, identified the gastro-intestinal tract as a site of long-term parasite persistence, revealed the infection dynamics of genetically modified parasites, and played a central role in Chagas disease drug development programmes. However, a major drawback of bioluminescence imaging is that it does not allow visualisation of host:parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualising T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterise their immunological context.

In Vivo Analysis of Trypanosoma cruzi Persistence Foci at Single-Cell Resolution

“…In some experiments, the colon was removed after standard imaging, an incision was made down the line of mesentery attachment, and the tissue was pinned out under a dissection microscope. Using ultrafine tweezers, large sections of the smooth muscular coat from the other layers were peeled off, while the tissue remained bathed in 0.3 mg/ml D᎑luciferin (41). After imaging, luciferin was removed by two washes with PBS.…”

Intracellular DNA replication and differentiation of Trypanosoma cruzi is asynchronous within individual host cells in vivo at all stages of infection

“…into each mouse (BALB/c or C3H/HeN) and the course of infection followed by BLI. At specific time-points, the mice were euthanised by an overdose of pentobarbital sodium and necropsied (for detailed description of the necropsy method, see [22]). Their organs were subject to post mortem BLI.…”

“…Tissue sections were produced as described previously [20,22]. Briefly, excised tissue was fixed in pre-chilled 95% ethanol for 20-24 hours in histology cassettes.…”

Motility patterns of Trypanosoma cruzi trypomastigotes correlate with the efficiency of parasite invasion in vitro

“…T. cruzi is able to infect a wide variety of nonprofessional phagocytic cells 18 , both in vitro 2,[35][36][37][38] and in vivo [39][40][41][42] . Although in the vertebrate host, the parasite can invade somatic cells in a wide range of tissues 43 , disease www.nature.com/scientificreports/ pathology occurs mostly in the heart and the digestive system.…”