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

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

Cerebral Malaria and Neuronal Implications of Plasmodium Falciparum Infection: From Mechanisms to Advanced Models

Abstract Reorganization of host red blood cells by the malaria parasite Plasmodium falciparum enables their sequestration via attachment to the microvasculature. This artificially increases the dwelling time of the infected red blood cells within inner organs such as the brain, which can lead to cerebral malaria. Cerebral malaria is the deadliest complication patients infected with P. falciparum can experience and still remains a major public health concern despite effective antimalarial therapies. Here, the current understanding of the effect of P. falciparum cytoadherence and their secreted proteins on structural features of the human blood‐brain barrier and their involvement in the pathogenesis of cerebral malaria are highlighted. Advanced 2D and 3D in vitro models are further assessed to study this devastating interaction between parasite and host. A better understanding of the molecular mechanisms leading to neuronal and cognitive deficits in cerebral malaria will be pivotal in devising new strategies to treat and prevent blood‐brain barrier dysfunction and subsequent neurological damage in patients with cerebral malaria

Fold changes in the expression of GSH peroxidase, superoxide dismutase and caspase-3 after the incubation of SH-SY5Y and U251 cells with pyrimethanil, cyprodinil and fludioxonil singly and in a mixture at a low (62.5 µM) and high (500 µM) exposure concentration over 48 h (n = 3 per determination; *, P<0.05).

<p>Fold changes in the expression of GSH peroxidase, superoxide dismutase and caspase-3 after the incubation of SH-SY5Y and U251 cells with pyrimethanil, cyprodinil and fludioxonil singly and in a mixture at a low (62.5 µM) and high (500 µM) exposure concentration over 48 h (n = 3 per determination; *, P<0.05).</p

Respective IC₅₀ values <b>(µM)</b> after exposure of U251 and SH-SY5Y cell lines to pyrimethanil, cyprodinil and fludioxonil singly and in combination using three viability assays (CellTiter Blue™, JC-1 and ATP).

<p>* P<0.05,</p>**<p>P<0.001,</p>***<p>P<0.001.</p>a)<p>denotes significant difference between single pesticide and combination IC50 values for each of the 3 assays.</p>b)<p>denotes significant difference between respective CellTiter Blue™ and JC-1 IC50 values.</p>c)<p>denotes significant difference between respective CellTiter Blue™ and ATP IC50 values.</p>@<p>denotes P<0.05.</p>&<p>denotes P<0.001.</p

GSH determinations expressed as a percentage of control values after the incubation of SH-SY5Y and U251 cells with pyrimethanil, cyprodinil and fludioxonil used singly and in combination, over 1–1000 µM over 48 h (n = 3 per determination; *, P<0.05; **, P<0.01; ***, P<0.001).

<p>GSH determinations expressed as a percentage of control values after the incubation of SH-SY5Y and U251 cells with pyrimethanil, cyprodinil and fludioxonil used singly and in combination, over 1–1000 µM over 48 h (n = 3 per determination; *, P<0.05; **, P<0.01; ***, P<0.001).</p