Fluorescent Trypanosoma cruzi parasites combined with in vivo imaging techniques facilitate testing for drug and vaccine efficacy

BUSTAMANTE JM, PADILLA AM, COLLINS MH, CANAVACI AM, XU D, PÉREZ BRANDÁN CM, TARLETON RL

Athens, Georgia, USA

Simposio; 19th Annual Molecular Parasitology/Vector Biology Symposium; 2009

Center for Tropical and Emerging Global Diseases

In vivo imaging techniques have opened the possibility of tracking labeled cells or pathogens in live animals.  Advantages of these methods include their non-invasive nature and amenability to frequent serial measurements.  Here we describe the use of Trypanosoma cruzi expressing fluorescent proteins  for in vitro and in vivo drug assays and vaccine protection studies.  We generated parasite lines expressing tandem tomato fluorescent protein (tdTomato) by transfection of T. cruzi epimastigotes with the pTREX-Neo-tdTomato plasmid.  These parasites are highly fluorescent as determined by flow cytometry and fluorescence microscopy.  For the in vitro drug assays, 104 parasites / well were plated in black 96 well plates with or without drug  and the change in fluorescence intensity was determined daily as a surrogate for growth.  For the in vivo drug testing, Balb/c mice were infected with 105 T. cruzi-tdTomato parasites in each hind footpad and treated or not with drug beginning at 6 days post-infection.  Parasite load at the site of infection was significantly diminished in the benznidazole (BNZ)-treated animals 3 days after the initiation of treatment.  Importantly, the efficacy of drugs in this short-term in vivo assay mirrored that of drugs administered over a 40 day course of treatment.  For vaccine protection studies, C57BL/6 mice were immunized by oral administration of metacyclic forms of T. cruzi parasites attenuated by mutations in the enoyl-CoA hydratase genes (ech1+/- ech2-/-).  Following immunization, mice were challenged with 2.5 x 105 T. cruzi-tdTomato parasites in each hind footpad.  Vaccine-induced protection was evident within the first week of infection.  Immunized mice displayed enhanced parasite clearance as shown by a reduced fluorescence signal at the site of infection as compared to non-immunized mice.  In conclusion, the development of T. cruzi-tdTomato parasites in combination with imaging technologies provides us with convenient methods for the in vitro and in vivo screening of new drugs as well as for the assessment of protection induced by vaccine candidates.