A new experimental model for assessing drug efficacy against Trypanosoma cruzi infection based on highly sensitive in vivo imaging

Abstract

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, one of the world's major neglected infections. Although development of improved antiparasitic drugs is considered a priority, there have been no significant treatment advances in the past 40 years. Factors that have limited progress include an incomplete understanding of pathogenesis, tissue tropism, and disease progression. In addition, in vivo models, which allow parasite burdens to be tracked throughout the chronic stage of infection, have been lacking. To address these issues, we have developed a highly sensitive in vivo imaging system based on bioluminescent T. cruzi, which express a red-shifted luciferase that emits light in the tissue-penetrating orange-red region of the spectrum. The exquisite sensitivity of this noninvasive murine model has been exploited to monitor parasite burden in real time throughout the chronic stage, has allowed the identification of the gastrointestinal tract as the major niche of long-term infection, and has demonstrated that chagasic heart disease can develop in the absence of locally persistent parasites. Here, we review the parameters of the imaging system and describe how this experimental model can be incorporated into drug development programs as a valuable tool for assessing efficacy against both acute and chronic T. cruzi infections.

Keywords: Chagas disease; drugs; imaging; trypanosomes.

Figure 1.

In vivo imaging of Trypanosoma cruzi. (A) The vector pTRIX2-RE9h was constructed by inserting the red-shifted firefly luciferase variant gene PpyRE9h into the T. cruzi rDNA targeting plasmid pTRIX. For transfection, the linearized targeting fragment can be produced by digesting the vector with the restriction enzymes indicated by arrows. (B) Cloned pTRIX2-RE9h–transfected T. cruzi epimastigotes, paraformaldehyde fixed and stained with anti–luciferase polyclonal antibody. Parasite DNA is stained red with Hoechst 33342. (C) Standard procedure for in vivo imaging experiments. Mice can be infected with bioluminescent parasites by the intraperitoneal, intravenous, or subcutaneous routes. At defined time points thereafter, they are inoculated with 150 mg^–1 kg^–1 d-luciferin, anaesthetized, and imaged for up to 5 min using an IVIS Lumina II system (PerkinElmer, Seer Green, UK). (D) Establishing the optimal “imaging window.” The variation in total ventral bioluminescence flux over time following injection of the luciferin substrate is shown.

Figure 2.

Highly sensitive in vivo imaging achieved with red-shifted luciferase. (A) Ventral images of SCID mice 1 h after intraperitoneal injection with bioluminescent bloodstream trypomastigotes (CL Brener strain, numbers indicated). The bioluminescence intensity is based on a log₁₀ scale heatmap as indicated (right). (B) Abdominal bioluminescence displayed by mice 1 h after infection with 100 or 1000 bloodstream trypomastigotes. The regions used for signal quantification are outlined by the red circles in A. (C) Quantification of abdominal bioluminescence for mice shown in A. Dotted line represents background +2 SD. Figure adapted from Lewis et al.

Figure 3.

Imaging the development of chronic Trypanosoma cruzi infections. (A) Ventral and dorsal images of the same individual BALB/c mouse infected with PpyRE9h-expressing T. cruzi imaged at the indicated day postinfection (dpi) over the course of >1 year. (B) Quantification of combined ventral and dorsal bioluminescence for the mouse shown in A. Gray line and dashed line are the mean and mean +2 SDs, respectively, for uninfected control mice. (C) Ex vivo bioluminescence analysis of selected organs and tissue samples imaged immediately postmortem. An example is shown for acute (14 dpi) and chronic (167 dpi) infection. 1, lung; 2, heart; 3, visceral adipose; 4, skeletal muscle (quadriceps); 5, spleen; 6, gut mesenteries; 7, stomach; 8, small intestine; 9, large intestine.

Figure 4.

Validation of in vivo imaging as a tool to assess efficacy of drug treatment against acute and chronic Trypanosoma cruzi infections. (A) SCID mice infected with 10³ bioluminescent T. cruzi bloodstream trypomastigotes (CL Brener strain) were treated (T) with benznidazole (daily by oral route, 100 mg^–1/kg^–1) 14 to 19 days postinfection (dpi) and compared with control nontreated (NT) mice. The bioluminescence intensity is represented by a log₁₀ scale heatmap as indicated (right). (B) BALB/c mice chronically infected with bioluminescent T. cruzi were treated (T) for 5 days with 100 mg^–1/kg^–1 benznidazole by the oral route starting at 66 dpi and compared with control nontreated (NT) mice. The bioluminescence intensity is represented by a linear scale heatmap as indicated (right). (C) Quantification of total ventral bioluminescence for the experiment including the mice shown in B (n = 3). Gray line and dashed line are the mean and mean +2 SDs, respectively, for uninfected control mice. Red arrows indicate treatment days.

Figure 5.

Combining in vivo imaging and immunosuppression to monitor the effectiveness of benznidazole treatment. Ventral views of BALB/c mice infected with 10³ bioluminescent Trypanosoma cruzi bloodstream trypomastigotes and treated with benznidazole. (A) Nontreated control. (B) Nontreated, immunosuppressed with 200 mg-¹ kg^-1 cyclophosphamide by intraperitoneal injection 124, 127, 130, and 133 days postinfection (dpi). (C) Treated with daily oral doses of 10 mg^-1 kg^-1 benznidazole between 90 and 110 dpi, then immunosuppressed with cyclophosphamide as above. (D) Treated with daily oral doses of 100 mg^-1 kg^-1 benznidazole between 90 and 110 dpi, then immunosuppressed with cyclophosphamide. The bioluminescence intensity is represented by a linear scale heatmap as indicated (left).