Naturally occurring triggers that induce apoptosis-like programmed cell death in Plasmodium berghei ookinetes

Abstract

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.

Figure 1. The effect of nitric oxide donors on the induction of apoptosis in P. berghei ookinetes.

Ookinetes were incubated in RPMI with or without the addition of SNP at different concentrations or for different time periods and then examined for the presence of different markers of apoptosis. A:The effect of 2 mM SNP on the proportion of ookinetes expressing a marker of apoptosis; data are means of 3 experiments (n = 3) with 300 ookinetes examined in each experiment. B,C: The effect of different concentrations of SNP following 1 h incubation; n = 3, with 100–150 ookinetes examined per experiment). D: The effect of different concentrations of SNP on ookinetes following 4 h incubation; n = 3, with 25–50 ookinetes examined in each experiment. Apoptosis markers examined were condensed nuclear chromatin (A, B), activated caspase-like molecules (C), and PS translocation without loss of membrane integrity (D). Error bars represent standard error of the mean (SEM). Bars with different letters are significantly different. White bars represent controls and patterned bars represent treated ookinetes.

Figure 2. Loss of mitochondrial membrane potential and the induction of apoptosis in P. berghei ookinetes.

Ookinetes were maintained in RPMI alone (controls) or incubated with 100 µM SNP for 2 h or with CCCP for 30 min prior to examination for loss of mitochondrial membrane potential (MOMP) (black bars) or the presence of nuclear chromatin condensation (grey bars). Error bars represent standard error of the mean (SEM), n = 4 with 50–100 ookinetes examined in each experiment. Bars with different letters are significantly different.

Figure 3. The effect of a nitric oxide synthase inhibitor on the expression of apoptosis markers by ookinetes in vivo.

An. stephensi were fed on blood containing P. berghei gametocytes (black bars) with the addition of L-NAME (grey bars) or the inactive isomer D-NAME (white bars). A: The proportion of ookinetes with condensed nuclear chromatin at 18 h and 20 h post-infection. B, C:The proportion of ookinetes expressing caspase-like activity or with compromised membranes (CM) 15 h and 20 h post-infection respectively. Error bars represent standard error of the mean (SEM). Bars with different letters are significantly different.

Figure 4. The effect of SNP on the survivorship of P. berghei-infected mosquitoes.

Four groups of 38–45 mosquitoes were fed on P. berghei-infected mouse blood (solid black lines, 2 groups), or P. berghei- infected mouse blood with the addition of 100 µM SNP (A) or 1 mM SNP (B) (dotted red lines). Mosquito deaths were recorded daily.

Figure 5. The effect of SNAP on the prevalence and intensity of P. berghei infection in An. gambiae.

Mosquitoes were fed on gametocytaemic mouse blood alone or on blood containing 100 µM SNAP and midguts examined 16–18 h post-infection. A: Prevalence of ookinete infections (mean of 5 experiments). B: Intensity of infection (the number of ookinetes recovered from each midgut; infected blood, n = 60, infected blood with SNAP, n = 55.

Figure 6. The effect of L-DOPA on the induction of apoptosis in P. berghei ookinetes in vitro.

A: Ookinetes were incubated in supplemented RPMI containing different concentration of L-DOPA for 1 h (bars with small dots), 2 h (bars with strips) and 4 h (bars with large dots) and the proportion containing condensed nuclear chromatin was calculated. B: Ookinetes were incubated with L-DOPA for 4 h and those with compromised membranes (dead ookinetes) were identified by staining with erythrosin B (grey bars), while ookinetes showing chromatin condensation were identified with acridine orange (white bars). Bars are a mean of 3 experiments (n = 3) with at least 50 ookinetes examined in each experiment. Error bars represent SEM.

Figure 7. The effect of H₂O₂ on the induction of apoptosis of ookinetes in P. berghei ookinetes in vitro.

The effect of incubation with different concentrations of H₂O₂ for 1 h (A) or 4 h (B). Apoptotic ookinetes displaying chromatin condensation were detected with acridine orange (white bars); ookinetes with compromised membranes were detected with propidium iodide (grey bars). Bars represent means of 3 experiments and at least 25 ookinetes were counted per sample. Error bars represent SEM and bars with different letters are significantly different.