In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds

Abstract

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

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).