Granular Formation during Apoptosis in Blastocystis sp. Exposed to Metronidazole (MTZ)

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.

Fig 1. Viable and apoptotic rate of untreated and treated forms of Blastocystis sp.

(a) viability rate and (d) apoptotic rate of untreated forms of Blastocystis sp. of st1, st2, st3 and st5. (b) viability rate and (e) apoptotic rate of treated forms of Blastocystis sp.(symptomatic isolates) at 0.0001mg/ml of st1, st2, st3 and st5. (c) viability rate and (f) apoptotic rate of treated forms of Blastocystis sp.at 0.01mg/ml of st1, st2, st3 and st5 (st = subtype).

Fig 2. Apoptotic and non-apoptotic granular forms of Blastocystis sp.

Light microscopy images showing drug treated Blastocystis sp. stained with tryphan blue to test the viability and Blastocystis sp. stained with Annexin (V)-FITC (green). Annexin V labeled with fluorescein (FITC) identifies apoptotic cells binding to PS exposed on the outer membrane of the cell.

Fig 3. Granular cells of the viable apoptotic (VA) viable non-apoptotic (VNA), non-viable apoptotic ((NVA) forms and necrosis forms.

The untreated cells are represented (a) subtype 3, (c) subtype 1 (e) subtype 2 and (g) subtype 5. The treated ones are represented by (b) subtype 3, (d) subtype 1, (f) subtype 2 and (h) subtype 5.

Fig 4. Mitochondrial transmembrane potential.

(a) and (d) granular cells viewed under bright field. (b) and (e) MitoCapture accumulates and aggregate in the mitochondria, giving off a bright red fluorescence (c)low intensity of green fluorescence indicating that the granular cells are still viable. (f) high intensity of green fluorescence indicating that the cells are dying (or apoptotic).

Fig 5. Accumulation of lipid droplets.

Presence of lipid in (a) viable non apoptotic (VNA) and (b) viable apoptotic (VA) (c) and (d) non-viable apoptotic (NVA) granular forms of Blastocystis sp.

Fig 6. Acridine orange and DAPI stained Blastocystis sp.

VA = Viable Apoptotic, NVA = Non- Viable Apoptotic, VNA = Viable Non Apoptotic.

Fig 7. Acridine orange stained granular forms.

(a) and (e) light microscopy images showing drug treated Blastocystis sp. (b) and (f) Blastocystis sp. stained with Annexin (V) (green) (c) and (g) Blastocystis sp. stained with acridine orange stain (orange). Annexin V labeled with fluorescein (FITC) identifies apoptotic cells binding to PS exposed on the outer membrane of the cell. Acridine orange stains the DNA of nucleus, mucus, and RNA as bright green, dull green and flaming red–orange, respectively. A = Apoptotic; NA = Non Apoptotic; LA = Late Apoptotic (Necrosis).

Fig 8. Different shades of acridine orange stained Blastocystis sp.

Blastocystis sp. of subtype 3 (st3) in treated (0.0001mg/ml) condition and images above the graphs show flaming red orange colouration of Blastocystis sp. at the 12 hours, decreasing gradually as the hours goes by and clarity of cells start increasing. At the 72 hour to 84 hour, progeny- like forms could be visualized.

Fig 9. TEM photographs.

(a), (c) and (e) shows untreated Blastocystis sp. whereas (b), (d) and (f) shows treated Blastocystis sp. (g) Electron dense granular forms of treated Blastocystis sp. (h) under high magnification; vacuolar form of Blastocystis sp. present within the granular forms. (CR = cristae).