Abstract
BackgroundThe options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage.Methodology/Principal FindingsWe therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D2, quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds.Conclusions/SignificanceParasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.
Highlights
IntroductionIn the absence of a vaccine, control of the lethal disease human African trypanosomiasis ( known as African sleeping sickness) relies on case treatment and elimination of the tsetse fly vector which carries the infecting trypanosomes
In the absence of a vaccine, control of the lethal disease human African trypanosomiasis relies on case treatment and elimination of the tsetse fly vector which carries the infecting trypanosomes
The search for new drugs is long and expensive, and determining which compounds are worth pursuing is a key challenge in that process
Summary
In the absence of a vaccine, control of the lethal disease human African trypanosomiasis ( known as African sleeping sickness) relies on case treatment and elimination of the tsetse fly vector which carries the infecting trypanosomes. For medium and high throughput screening of the pathogenic bloodstream form (BF) of T. brucei, recent studies have used ATP levels in the culture as a barometer of parasite growth inhibition [2]. Another assay that has been used relies on the reduction of Alamar blue (resazurin) to a red fluorescent compound in live cells [3,4]. The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage
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