Oxidative stress-induced cell cycle blockage and a protease-independent programmed cell death in microaerophilic Giardia lamblia

Abstract

Giardia lamblia is a microaerophilic human gastrointestinal parasite and considered as an early-diverged eukaryote. In vitro oxidative stress generation plays a significant role in cell cycle progression and cell death of this parasite. In the present study hydrogen peroxide, metronidazole, and a modified growth medium without cysteine and ascorbic acid have been chosen as oxidative stress-inducing agents. Cell cycle progression has been found to be regulated by different types of oxidative stresses. Apoptosis is not an established pathway in Giardia, which is devoid of ideal mitochondria, but in the present investigation, apoptosis-like programmed cell death has been found by the experiments like AnnexinV-FITC assay, DNA fragmentation pattern, etc. On the contrary, Caspase-9 assay, which confirms the caspase-mediated apoptotic pathway, has been found to be negative in all the stress conditions. Protease inhibitor assay confirmed that, even in absence of any proteases, programmed cell death does occur in this primitive eukaryote. All these results signify a novel pathway of programmed suicidal death in Giardia lamblia under oxidative stress. This is the first demonstration of protease-independent programmed cell death regulation in Giardia exclusive for its own specialties.

Keywords: Giardia lamblia; apoptosis; early branching eukaryotes; oxidative stress; programmed cell death; reactive oxygen species.

Figure 1

Cell cycle analysis of control and treated cells. D, E, and F are showing sub-G1*, G1 and G2/M phase in the trophozoite cell cycle. Each unit distance in the X-axis denotes 2N DNA content of the trophozoites. Normal Giardia trophozoite contains 4N genomic DNA content in G1 phase. i) In the control set, maximum no. of trophozoites are showing 4N DNA content with few sub G1 population indicating apoptotic cell death. ii) Metronidazole treated cells stop their cell cycle propagation just at synthesis phase and commit apoptotic type of cell death. Cells lie in between D to late E region. iii) Effect of H₂O₂ on Giardia trophozoites is very prompt. All the trophozoites undergo apoptotic type of death and lie sharply in the D region, sharply within sub G1 stage. iv) Cells treated with modified medium show a group of trophozoites in D region with sub-G1 phase, but few cells are moving beyond S phase towards G2, indicating a tendency to encystation in stressed condition. Notes: ^*Sub-G1 implies that the DNA content is less than usual G1 stage (4N in case of Giardia). In the dead cells when nuclei start degradation then another peak arises in flow cytometer just left of the original G1 peak, indicating that the DNA content is decreasing.

Figure 2

Electron microscopic view of control and treated cells. A) Live Giardia cell with two nuclei and flagella. B) Live Giardia (mainly two nuclei are focused). C) Giardia cells treated with H₂O₂: Nuclear membrane blebbing due to PCD. D) Giardia cells treated with metronidazole: Increased vacuolation and outer membrane blebbing due to PCD. E and F) Giardia cells treated with modified medium: Increased vacuolation and nuclear membrane and outer membrane blebbing due to PCD. G) Change in cell morphology after treated with modified medium. Abbreviations: N, nucleus; P, plasma membrane.

Figure 3

Annexin V-FITC assay. A) normal live Giardia cells, B) Giardia cells treated with H₂O₂ for 2 h. C) Giardia cells treated with protease inhibitor cocktail and then further treated with H₂O₂ for 2 h. D) Giardia cells treated with metronidazole for 4 h. E) Giardia cells treated with protease inhibitor cocktail and then further treated with metronidazole for 4 h. F) Giardia cells grown in modified medium deprived of Cys-ascorbate for 6 h. G) Giardia cells grown in modified medium deprived of Cys-ascorbate with protease inhibitor cocktail. Panels B to G clearly show ring formation due to apoptosis.

Figure 4

Flow cytometric analysis of FITC-PI stained cells. A) Control live cells lying in the left hand bottom quadrant (B3) as the cell population has not taken any of the two dyes, FITC or PI. B) H₂O₂ treated cells after one hour takes FITC and lie in the right hand bottom quadrant (B4) indicating apoptotic type of death. Similarly, in C) and D) cells treated with metronidazole and modified medium respectively, show their mode of death in apoptotic way.

Figure 5

Degradation of nuclei due to programmed cell death in Giardia. A) normal Giardia cells PI stained, B) Giardia cells treated with H₂O₂ for 2 h and PI stained. C) Giardia cells treated with protease inhibitor cocktail and then further treated with H₂O₂ for 2 h and PI stained. D) Giardia cells treated with metronidazole for 4 h and PI stained. E) Giardia cells treated with protease inhibitor cocktail and then further treated with metronidazole for 4 h and PI stained. F) Giardia cells grown in modified medium deprived of Cys-ascorbate for 4 h and PI stained. G) Giardia cells grown in modified medium deprived of Cys-ascorbate with protease inhibitor cocktail and PI stained. Panels B to G clearly show degradation of nuclear content. The phase images in all seven pictures are shown in the right side of the individual pictures.

Figure 6

DNA fragmentation assay. 1st panel (C) from left is indicating the DNA from the control live cells. A distinct DNA band can be observed here. In the 2nd panel (CA) fragmented sheared DNA (mainly in lower molecular weight region) was observed in case of modified medium. Similarly in the third and fourth panel (H and M) for hydrogen peroxide and metronidazole respectively, DNA fragmentation can be observed. The DNA marker ranges from 1–10 kb.