Abstract
The role and function of the granular life cycle stage in Blastocystis sp, remains uncertain despite suggestions being made that the granules are metabolic, reproductive and lipid in nature. This present study aims to understand granular formation by triggering apoptosis in Blastocystis sp. by treating them with metronidazole (MTZ). Blastocystis sp.cultures of 4 sub-types namely 1, 2, 3 and 5 when treated with 0.01 and 0.0001 mg/ml of metronidazole (MTZ) respectively showed many of the parasites to be both viable and apoptotic (VA). Treated subtype 3 isolates exhibited the highest number of granular forms i.e. 88% (p<0.001) (0.0001 mg/ml) and 69% (p<0.01) (0.01 mg/ml) respectively at the 72 h in in vitro culture compared to other subtypes. These VA forms showed distinct granules using acridine orange (AO) and 4’,6-diamino-2-phenylindole (DAPI) staining with a mean per cell ranging from 5 in ST 5 to as high as 16 in ST 3. These forms showed intact mitochondria in both viable apoptotic (VA) and viable non-apoptotic (VNA) cells with a pattern of accumulation of lipid droplets corresponding to viable cells. Granular VA forms looked ultra-structurally different with prominent presence of mitochondria-like organelle (MLO) and a changed mitochondrial trans-membrane potential with thicker membrane and a highly convoluted inner membrane than the less dense non-viable apoptotic (NVA) cells. This suggests that granular formation during apoptosis is a self-regulatory mechanism to produce higher number of viable cells in response to treatment. This study directs the need to search novel chemotherapeutic approaches by incorporating these findings when developing drugs against the emerging Blastocystis sp. infections.
Highlights
Blastocystis sp. is an intestinal parasite with polymorphic forms such as vacuolar, granular, amoeboid[1,2] cystic, avacuolar, and multi-vacuolar forms [3,4] the most commonly detected forms in stools being the vacuolar form
In the present study by correlating the dynamics of granular formation within the various subtypes of Blastocystis sp., to apoptosis caused by treatment with metronidazole, we provide evidence that when a cell is triggered to undergo apoptosis, granular forms play a crucial role in continuing the propagation of Blastocystis sp
We have reported that apoptosis in Blastocystis sp. occurs via caspase dependent and a smaller percentages of cells undergo caspase independent pathways [33]
Summary
Blastocystis sp. is an intestinal parasite with polymorphic forms such as vacuolar, granular, amoeboid[1,2] cystic, avacuolar, and multi-vacuolar forms [3,4] the most commonly detected forms in stools being the vacuolar form. Seen growing despite the short period in, in vitro cultures suggested that the organism must be having another asexual mode of reproduction other than binary fission implicating these granules to be reproductive [2,12,13] This was refuted the reason being that the suggested progeny forms showed striking similar characteristics with the metabolic granules described previously as well as the lack of evidence for these to be viable [1,14,15] There was a postulation that the granules is a result of a mechanism trigger for apoptotic body deposition in the central body in Blastocystis sp. The intracellular granules have been suggested to be heterogeneous and have been described as being myelin-like inclusions, small vesicles, crystalline granules, and lipid droplets [17]
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