Figure 1.
Plasmids used in the generation of fluorescent and bioluminescent T. cruzi.
Schematic representation of (A) the pTrex-Neo-tdTomato plasmid, and (B) the pLew90β-GW/T7/PARP SAS/luciferase/Aldolase 3′UTR plasmid used for generation of the T. cruzi reporter lines.
Figure 2.
Fluorescence evaluation of tdTomato parasites.
(A) Microscope image showing the tdTomato expressing parasites in all the life stages. (B) The fluorescence intensity in epimastigotes was assessed using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). No decrease in fluorescence intensity was observed in parasites cultured for >5 months with (red) and without antibiotic (blue). The background fluorescence of WT parasites (green) can also be observed.
Figure 3.
In vitro epimastigote growth assays using tdTomato parasites.
(A) Epimastigotes growth over time in the presence of benznidazole at the indicated concentrations and comparison of measurement of drug inhibition of epimastigote growth by fluorescence and visual counting by hemacytometer. (B) Intra-assay analysis (left) showing the low variation among wells with the same drug concentration (n = 4). Inter-assay analysis (right) showing the low variation among IC50 curves from individual assays. (C) IC50 calculation in response to benznidazole and the EXO2 derivatives activity against epimastigotes after 3 days of treatment/culture.
Figure 4.
In vitro amastigote growth assays using tdTomato parasites.
(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of T. cruzi expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).
Figure 5.
Fluorescent T. cruzi-tdTomato expressing parasites imaged post-treatment.
Mice (10 per group) were infected in the hind foot pads with 2.5×105 T. cruzi tdTomato trypomastigotes and the images were taken every two days from day 1 to 11 post infection. (A) Images from days 5, 7 and 9 post infection. (B) Quantification of the fluorescent signal from mice in panel A at all imaging points.
Figure 6.
Luminescent T. cruzi imaged at various times post-treatment.
(A) Mice (10 per group) were infected in the footpad with 1×105 T. cruzi bioluminescent trypomastigotes. For all images shown the color scale ranges from blue (with a minimum set at 60 photons/s/cm2/sr) to red (maximum of 3000 photons/s/cm2/sr). (B) Quantification of luminescent signal from mice in panel A.
Figure 7.
Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.
(A) Evolution of parasitemia after infection with 1×103 CL strain of T. cruzi on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).