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. 2023 Feb 14;11(2):476.
doi: 10.3390/microorganisms11020476.

Impact of Laboratory-Adapted Intracellular Trypanosoma cruzi Strains on the Activity Profiles of Compounds with Anti- T. cruzi Activity

Melissa L Sykes  1   2 , Emily K Kennedy  1 , Vicky M Avery  1   2
Affiliations

Impact of Laboratory-Adapted Intracellular Trypanosoma cruzi Strains on the Activity Profiles of Compounds with Anti- T. cruzi Activity

Melissa L Sykes et al. Microorganisms. .

Abstract

Chagas disease is caused by infection with the protozoan parasite, Trypanosoma cruzi. The disease causes ~12,000 deaths annually and is one of the world's 20 neglected tropical diseases, as defined by the World Health Organisation. The drug discovery pipeline for Chagas disease currently has few new clinical candidates, with high attrition rates an ongoing issue. To determine if the Trypanosoma cruzi strain utilised to assess in vitro compound activity impacts activity, a comparison of laboratory-adapted T. cruzi strains from differing geographical locations was undertaken for a selection of compounds with anti-T. cruzi activity. To minimise the possible effect of differences in experimental methodology, the same host cell and multiplicity of infection were utilised. To determine whether the compound exposure time influenced results, activity was determined following exposure for 48 and 72 h of incubation. To ascertain whether replication rates affected outcomes, comparative rates of replication of the T. cruzi strains were investigated, using the nucleoside analogue, 5-ethynyl-2'-deoxyuridine. Minimal differences in the in vitro activity of compounds between strains were observed following 48 h incubation, whereas significant differences were observed following 72 h incubation, in particular for the cytochrome P450 inhibitors tested and the cell cycle inhibitor, camptothecin. Thus, the use of panels of laboratory adapted strains in vitro may be dependent on the speed of action that is prioritised. For the identification of fast-acting compounds, an initial shorter duration assay using a single strain may be used. A longer incubation to identify compound activity may alternatively require profiling of compounds against multiple T. cruzi strains.

Keywords: Chagas disease; Trypanosoma cruzi; compound activity; drug discovery; image-based assay; parasite strains.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Emax values of compounds from differing chemical classes against T. cruzi intracellular amastigotes, from Tulahuen, CL, Y and G strain parasites, following 48 h incubation. (A) CYP51 inhibitors (B) camptothecin, clemastine fumarate and two compounds from the MMV Pathogen Box collection. (C) compounds which cleared 100% of parasites from host cells over all strains: the nitroheterocyclic inhibitors benznidazole and nifurtimox; and the antifungal, ciclopirox olamine. Solid bars = Tulahuen strain parasite, dotted bars = CL strain, chequered bars = Y strain, striped bars = G strain. Dashes shown at 80% and 100% activity.
Figure 2
Figure 2
Emax values of compounds from differing chemical classes against T. cruzi intracellular amastigotes, from Tulahuen, CL, Y and G strain parasites, following 72 h incubation. (A) CYP51 inhibitors (B) camptothecin, clemastine fumarate and two compounds from the MMV Pathogen Box collection. (C) compounds which cleared 100% of parasites from host cells over all strains: the nitroheterocyclic inhibitors benznidazole and nifurtimox; and the antifungal, ciclopirox olamine. Solid bars = Tulahuen strain parasite, dotted bars = CL strain, chequered bars = Y strain, striped bars = G strain. Dashes shown at 90% and 100% activity.
Figure 3
Figure 3
Images of 3T3 infected cells stained with the nuclear dye Hoechst, following treatment with 3.7 µM of the eukaryotic cell cycle inhibitor, camptothecin. Cells infected with: (A) Tulahuen strain (B) CL strain (C) G strain and (D) Y strain. The nucleus of parasites and host cells stained with Hoechst are shown in blue, demonstrated in (A), inset, where P = parasite nuclei, H = host cell nucleus. A mean of 19.4 ± 2.3% more infected cells was observed for the G and Y strains, in comparison to Tulahuen and CL strains, following treatment. Images taken at 20× magnification using a Phenix (PerkinElmer) imaging system.
Figure 4
Figure 4
Intracellular replication of four T. cruzi strains, detected with the nucleoside analogue 5-ethynyl-2’-deoxyuridine following 48 and 72 h incubation. (A) The percentage of replicating intracellular parasites following 48 h (green) and 72 h incubation (blue). (B) Hoechst identification of intracellular Tulahuen parasites, following 72 h incubation (C) EdU identification of replicating Tulahuen parasite from the same cells as shown in (A). In (B,C) the image inset shows the difference in Hoechst and EdU identified parasites within a cell. Images taken at 20× magnification on a Phenix imaging system (PerkinElmer).
Figure 5
Figure 5
The number of host cells/well (A) and number of infected cells/well (B) following 48 h (green bars) and 72 h incubation (blue bars) with T. cruzi G, CL, Y and G strain parasites, respectively. Data taken from images acquired on a Phenix imaging system (PerkinElmer) and analysed using Harmony 4.8.
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