Abstract
BackgroundBiochemical evidence of a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, including Blastocystis spp. The distinct differences in the phenotypic characterization reported previously have prompted us to compare the rate of apoptosis in Blastocystis spp. isolated from individuals who were symptomatic and asymptomatic. In the current study, we analysed the caspase activation involved in PCD mediated by a cytotoxic drug, (metronidazole) in both symptomatic & asymptomatic isolates.MethodsApoptosis was induced in Blastocystis spp. by treating cultures of symptomatic and asymptomatic isolates of 3 sub-types namely 1, 3 and 5 with two different concentrations, 0.1 and 0.0001 mg/ml of metronidazole (with and without pre-treatment with a pan-caspase inhibitor, zVAD.fmk). The experiment was repeated to assess the number of apoptotic cells in all the isolates of both conditions.ResultsSymptomatic isolates of subtype 3 (without pre-treatment with a pan-caspase inhibitor, zVAD.fmk) showed high fluorescence intensity for active caspase-like proteases [0.0001 mg/ml, 88% (p < 0.001) at 0.1 mg/ml, 70% (p < 0.001)] at the 72nd hour in vitro culture in comparison with asymptomatic isolates [0.0001 mg/ml, 65%, at 0.1 mg/ml, 55%]. The number of apoptotic cells was higher [0.0001 mg/ml, 89% (p < 0.001) and at 0.1 mg/ml, 70% (p < 0.001)] at the 72nd hour of in vitro culture in comparison with asymptomatic isolates [0.0001 mg/ml, 66% (p < 0.001) and at 0.1 mg/ml, 45% (p < 0.01)]. Cells treated with metronidazole in the presence of zVAD.fmk showed less than 10% caspase activation.ConclusionThe high number of symptomatic cells expressing active caspase-like proteases and becoming apoptotic compared to asymptomatic cells clearly demonstrates that the response to metronidazole treatment is isolate dependent. Hence this justifies the conflicting reports on the curative success rates when treated with this drug. The study has also created a need to identify apoptosis effectors in Blastocystis spp of different isolates especially as it was shown that apoptosis was sub-typed related. These findings can be exploited for the development of diagnostic markers and novel therapeutic drugs to enhance the effectiveness of the diagnosis and treatment of the patients infected with Blastocystis spp.
Highlights
Biochemical evidence of a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, including Blastocystis spp
This caspase-independent pathway was referred to as a mitochondrial pathway. This pathway involves mitochondrial dysregulation resulting from a loss of mitochondrial membrane potential and release of pro-apoptotic proteins leading to cell death
Cells expressing caspase-like activity Caspase-like activation, an early event in apoptosis, was detected in all the symptomatic and asymptomatic isolates of both conditions [Figure 1a-d)]
Summary
Biochemical evidence of a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, including Blastocystis spp. Cell death occurrences were reported in the presence of the pan-caspase inhibitor zVAD.fmk, indicating the existence of caspase independent death effectors [2]. In such studies, this caspase-independent pathway was referred to as a mitochondrial pathway. Mitochondrial pro-apoptotic proteins were shown to be involved in caspase independent pathways [3] Both caspase-3like activation and mitochondrial dysregulation have been reported to occur during apoptosis in Blastocystis spp. the same study reported that, both pathways, when blocked, could not save the parasite from death [4]
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