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Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

Akide-Ndunge OB, Tambini E, Giribaldi G, McMillan PJ, Müller S, Arese P, Turrini F - Malar. J. (2009)

Bottom Line: Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent.Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs.Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino Medical School, Torino, Italy. oskar.akide@unito.it

ABSTRACT

Background: Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy.

Methods: Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane.

Results: In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites.

Conclusion: Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

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Stage-dependent mRNA expression of parasite antioxidant enzymes (A; B) and HSPs (C) in parasites growing in G6PD-deficient (Mediterranean variant) RBCs. Changes in mRNA expression were measured using qRT-PCR. For details, see legend to Figure 1 and Materials and methods. The results are presented as -fold increase over the mRNA level at early ring stage measured 13 h after reinfection using the 2-ΔΔCt method. Mean values ± SEM, vertical bars, n = 5–7. Significances vs ring stage levels are indicated with symbols p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).
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Figure 3: Stage-dependent mRNA expression of parasite antioxidant enzymes (A; B) and HSPs (C) in parasites growing in G6PD-deficient (Mediterranean variant) RBCs. Changes in mRNA expression were measured using qRT-PCR. For details, see legend to Figure 1 and Materials and methods. The results are presented as -fold increase over the mRNA level at early ring stage measured 13 h after reinfection using the 2-ΔΔCt method. Mean values ± SEM, vertical bars, n = 5–7. Significances vs ring stage levels are indicated with symbols p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).

Mentions: The effect of parasite growth in G6PD-deficient RBCs on the stage-dependent mRNA expression of parasitic antioxidant enzymes and HSPs is shown in Figure 3. The mRNA expression pattern of parasitic antioxidant enzymes and HSPs was also modulated stage-dependently and all considered parameters significantly increased in the ring-to-trophozoite transition: GPx-like TPx (6.1-fold), GR (12.8-fold), Grx (6.0-fold), GST (5.6-fold), 1-Cys-Prx (4.1-fold), SOD-1 and SOD-2 (3.7-fold), G6PD (2.6-fold), HSP60 (8.5-fold), HSP70–2 (7.5-fold), HSP70–3 (4.7-fold), HSP75 (15.8-fold), and HSP90 (6.1-fold). The kinetics of stage-dependent mRNA expression was apparently not affected by the G6PD-deficient status of the host RBCs, as gene expression of all parameters peaked at 19 h after reinfection and declined afterwards.


Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

Akide-Ndunge OB, Tambini E, Giribaldi G, McMillan PJ, Müller S, Arese P, Turrini F - Malar. J. (2009)

Stage-dependent mRNA expression of parasite antioxidant enzymes (A; B) and HSPs (C) in parasites growing in G6PD-deficient (Mediterranean variant) RBCs. Changes in mRNA expression were measured using qRT-PCR. For details, see legend to Figure 1 and Materials and methods. The results are presented as -fold increase over the mRNA level at early ring stage measured 13 h after reinfection using the 2-ΔΔCt method. Mean values ± SEM, vertical bars, n = 5–7. Significances vs ring stage levels are indicated with symbols p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2696464&req=5

Figure 3: Stage-dependent mRNA expression of parasite antioxidant enzymes (A; B) and HSPs (C) in parasites growing in G6PD-deficient (Mediterranean variant) RBCs. Changes in mRNA expression were measured using qRT-PCR. For details, see legend to Figure 1 and Materials and methods. The results are presented as -fold increase over the mRNA level at early ring stage measured 13 h after reinfection using the 2-ΔΔCt method. Mean values ± SEM, vertical bars, n = 5–7. Significances vs ring stage levels are indicated with symbols p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).
Mentions: The effect of parasite growth in G6PD-deficient RBCs on the stage-dependent mRNA expression of parasitic antioxidant enzymes and HSPs is shown in Figure 3. The mRNA expression pattern of parasitic antioxidant enzymes and HSPs was also modulated stage-dependently and all considered parameters significantly increased in the ring-to-trophozoite transition: GPx-like TPx (6.1-fold), GR (12.8-fold), Grx (6.0-fold), GST (5.6-fold), 1-Cys-Prx (4.1-fold), SOD-1 and SOD-2 (3.7-fold), G6PD (2.6-fold), HSP60 (8.5-fold), HSP70–2 (7.5-fold), HSP70–3 (4.7-fold), HSP75 (15.8-fold), and HSP90 (6.1-fold). The kinetics of stage-dependent mRNA expression was apparently not affected by the G6PD-deficient status of the host RBCs, as gene expression of all parameters peaked at 19 h after reinfection and declined afterwards.

Bottom Line: Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent.Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs.Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino Medical School, Torino, Italy. oskar.akide@unito.it

ABSTRACT

Background: Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy.

Methods: Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane.

Results: In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites.

Conclusion: Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

Show MeSH
Related in: MedlinePlus