Phenotypic and transcriptomic analyses of Plasmodium falciparum protein kinase A catalytic subunit inhibition

Abstract

The emergence and dissemination of drug-resistant malaria parasites represent one of the most important problems in malaria case management. Plasmodium falciparum is the causative agent of the most lethal form of human malaria. The molecular mechanisms that control the life cycle of the malaria parasite are still poorly understood. The published genome sequence (P. falciparum strain 3D7) reveals that several homologs of eukaryotic signaling proteins, such as protein kinases and phosphatases, are conserved in P. falciparum. Proteins kinases are now widely recognized as valuable drug targets in protozoan parasites. In this study, gene silencing with double-stranded RNA (dsRNA) and microarray techniques were used to study the biological function of the cAMP-dependent protein kinase catalytic subunit (PfPKAc) in the parasite erythrocytic life cycle. Treatment of parasites with PfPKAc dsRNA resulted in a marked reduction of endogenous PfPKAc mRNA associated with a compensatory decrease of PfPKAr mRNA followed by morphological changes in schizont stages and cell cycle arrest. The global effects of gene silencing were also investigated using a P. falciparum pan-genomic microarray. Transcriptomic analysis showed that the expression of 329 genes was altered in response to downregulation of PfPKAc mRNA particularly genes in specific metabolic pathways linked with merozoite invasion processes, the calcium/calmodulin signaling, and kinases network and mitochondrial functions.

Plasmodium falciparum proteome changes in response to doxycycline treatment

“…Real-time transcript quantification was performed using a 7900HT Fast Real-Time PCR system (Applied Biosystems). Amplification reactions and the 2 -ΔΔCt method of relative quantification to estimate relative expression of mRNA targets were performed as previously described [35]. All data were expressed as means ± standard deviation.…”

Evaluation of Five Antischizophrenic Agents Against Toxoplasma gondii in Human Cell Cultures

“…Calcium binding activates calmodulin and downstream calmodulin-dependent protein kinases have been implicated in life-cycle and erythrocyte invasion of the apicomplexan, Plasmodium falciparum (Vaid et al, 2008;Wurtz et al, 2009), but these pathways are not well understood in T. gondii. In our experiments, tachyzoites were observed within parasitophorous vesicles in host cells suggesting that antipsychotics might blunt signals for T. gondii egress.…”

cAMP-dependent protein kinase fromPlasmodium falciparum: an update

“…The expression of 6 reticulocytebinding-like (RBL) homologue genes was found to be down-regulated in response to pfpkac mRNA inhibition (Wurtz et al 2009a). These genes belong to the invasion/motility pathway and seem to be important for P. falciparum invasion of human erythrocytes (Tham et al 2009).…”

“…Many nuclear genes with mitochondrial signal sequences were found to be induced when pfpkac expression was inhibited (Wurtz et al 2009a). It is well known that parasite mitochondrial activity requires the import of many proteins (van Dooren et al 2006;TorrentinoMadamet et al 2009).…”