Abstract
Diseases caused by trypanosomatid parasites remain a significant unmet medical need for millions of people globally. Trypanosomatid parasites such as Trypanosoma cruzi and subspecies of Trypanosoma brucei cause Chagas disease and human African trypanosomiasis (HAT), respectively. Although efforts to find novel treatments have been successful for HAT, Chagas disease is still treated with decades-old therapies that suffer from long treatment durations and severe safety concerns. We recently described the identification and characterization of the cyanotriazole compound class that kills trypanosomes, in vitro and in vivo, by selective inhibition of the trypanosome nuclear topoisomerase II enzyme. To evaluate whether inhibition of the topoisomerase II enzyme led to parasite death due to lethal double-strand DNA breaks, we developed assays for detecting DNA damage in both intracellular amastigotes of T. cruzi and bloodstream-form T. brucei by using the canonical DNA damage marker γH2A. Herein, this article describes the protocols for detecting DNA damage using an immunofluorescence assessment of γH2A by microscopy in trypanosome parasites. Key features • Immunofluorescence-based assay to detect the γH2A response in T. brucei and T. cruzi parasites. • Robust DNA damage pathway-based cellular assays to evaluate topoisomerase II poisons' ability to cause DNA damage. • A 384-well plate-based T. cruzi protocol allows high-resolution and high-throughput evaluation of compounds that cause DNA damage by measuring γH2A in intracellular parasites. • This assay could be modifiable for evaluation of DNA damage responses in various intracellular and extracellular eukaryotic pathogens.
Keywords:
Chagas; Cyanotriazoles; DNA damage; Double-strand DNA break; Drug discovery; HAT; Histones; Immunofluorescence assay; Parasitology; Topo II poison; Topoisomerase II; Trypanosoma brucei; Trypanosoma cruzi; Trypanosomes; γH2A.