Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei

Abstract

Background: The 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 findings: We 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 D(2), 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/significance: Parasites 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.

Figure 1. Analysis of BF viability by flow cytometry.

A) On the left are untreated cells and on the right are cells treated with pentamidine. The top panels show staining with ethidium (dead cells are permeable to the dye) and calcein (live cells hydrolyze the fluorogenic substrate). The bottom panels show the forward and side scatter, with gates drawn to indicate the region containing live cells. B) The pentamidine-treated cells were divided into two populations by forward/side scatter as shown in Figure 1A and analyzed for calcein/ethidium staining.

Figure 2. Assays of mitochondrial potential and generation of ROS in drug-treated parasites.

A) Mitochondrial potential. After 24 hour drug treatment, parasites were incubated with rhodamine 123 and analyzed by flow cytometry. Dead cells, which were present in all samples, were excluded by gating on scatter. The arrow marks the midpoint for the untreated control. The legend includes the results from multiple experiments, showing the percentage decrease in number of live cells, average and standard deviation of rhodamine 123 fluorescence expressed as the ratio of the geometric means of the treated samples versus the untreated sample. B) Generation of ROS. Amount of ROS above that seen in untreated cells following a two-hour exposure to the indicated drugs at two different concentrations (see Table 1). ROS were assayed by flow cytometric detection of oxidized CM-H2DCFDA.

Figure 3. DNA content and genome segregation following drug treatment.

A) DNA content following 24 hour treatment revealed by PI staining of RNAse treated BF. The large peak at left seen in some samples represents cells with degraded DNA. 2C indicates diploid (G1) DNA content, 4C indicates G2/M DNA content. Note the appearance of cells with sub-G1 DNA (peaks at far left) following some treatments, as well as cells with higher order DNA content. In each case, the total number of live cells was less than 50% of the untreated controls. The percentage of dead cells (%†) is indicated on each graph. B) Duplication and segregation of the nucleus and kinetoplast following drug treatment. The same populations of cells analyzed in Figure 3A were subjected to microscopic analysis, enumerating the number of nuclei and kinetoplasts per cell as revealed by DAPI staining. Forms seen during normal cell cycle progression are indicated by blue shades, while red to yellow shades indicate abnormal, dead end forms. N, nucleus, K, kinetoplast.

Figure 4. Annexin V staining of populations with dying cells.

A) Annexin V and PI staining of unfixed cells. Untreated cells show predominantly annexin V-negative, PI-negative staining (lower left quadrant). All drug treated cells showed additional populations with higher annexin staining coupled with low to high PI staining. B) Annexin-positive cells are dead. The pentamidine-treated population shown in A was analyzed according to light scattering properties. The low forward scatter, dead population (D) contained all annexin-stained cells, including those with low to high PI staining. The live population contained the annexin-negative, PI-negative cells.

Figure 5. Representative growth curves for RNAi cell lines targeting TOPIBL, NOPP44/46, or TOPIImt.

Tet was added at day 0 to initiate destruction of the targeted RNA. The RNAi-induced cells from the final day of the growth curve were used in the experiments shown in Figure 6. The percent of dead cells at that point is listed. NOPP44/46 protein levels were reduced 44% as shown by immunoblotting (unpublished results).

Figure 6. DNA content and genome segregation following genetic knockdown.

The same parasites described in Table 4 were analyzed for DNA content (Panel A) and nuclear and kinetoplast genome segregation (Panel B) as described in Figure 3. The cumulative number of live cells was between 10% and 50% of control uninduced populations on the day assayed.