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.
Highlights
There is a well established body of literature that recognizes programmed cell death (PCD) as a signal dependent, active process of cell suicide that occurs in unicellular eukaryotes as well as metazoans
In order to determine whether the removal of these cells from gametocytaemic blood prior to culture affected the proportion of ookinetes that exhibited signs of chromatin condensation, a comparison was made between blood with and without White blood cells (WBCs)
An investigation into the effect of different concentrations of sodium nitroprusside (SNP) showed that the effect was dose dependent; 10 mM SNP did not induce additional ookinetes to undergo chromatin condensation, 100 mM SNP caused a significant increase of 30% (P,0.001) and 500 mM SNP caused an increase of 32% (P,0.001) (Figure 1B)
Summary
There is a well established body of literature that recognizes programmed cell death (PCD) as a signal dependent, active process of cell suicide that occurs in unicellular eukaryotes as well as metazoans. Three types of PCD have been well characterized in metazoans, including type 1 PCD, or apoptosis [6,7] it is recognized that overlapping pathways leading to different types of PCD death exist, and more complex nomenclature has been suggested [8]. Most of the features that are signatures of apoptosis in metazoans are present in unicellular eukaryotes, including loss of mitochondrial outer membrane permeability (MOMP), nuclear chromatin condensation, cytochrome c release, DNA fragmentation and translocation of phosphatidylserine (PS) to the outer cell membrane (reviewed in [10,11]). Much of the apoptotic molecular machinery common to multicellular eukaryotes is absent, and different pathways may be leading to the same endpoint [11,12,13]
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