Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of a double knockout of TCA cycle enzymes IDH and KDH

ABSTRACT: New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Although mitochondrial metabolism is the target of both existing drugs and new lead compounds, the role of the mitochondrial tricarboxylic acid (TCA) cycle remains poorly understood. Herein, we describe 11 genetic knockout parasite lines that delete six of the eight TCA cycle enzymes. Although all TCA knockouts grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase knockout parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass-spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development . Two parallel timecourses resulting in a total of 16 samples (8 wildtype, Isocitrate Dehydrogenase/alpha-Ketogluterate Dehydrogenase double knockout) were hybridized against a Cy3-labeled reference pool of 3D7 mixed stage parasites on a two-color array.

ORGANISM(S): Plasmodium falciparum

SUBMITTER: Heather Painter

PROVIDER: E-GEOD-59015 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programs around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline based drugs is becoming critical. So far only few resistance markers have been identified and verified from which only two ABC transmembrane transporters namely PfMDR1 and PfCRT have been experimentally verified. Another P. falciparum ABC transporter, the multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identify a parasite clone that is derived from the 3D7 P. falciparum strain and which shows increased resistance to chloroquine and mefloquine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5M-bM-^@M-^Y upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription that result in increased levels of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of such genetic polymorphisms to underlie drug resistance phenotypes. Presented here are the data from microarray-based genome-wide transcriptomic and genomic studies of the drug-sensitive and drug-resistant 3D7 clones 11C/wt and 6A/mut. 2 P. falciparum lab clones derived from 3D7 strain were harvested during the intra-erythrocytic cycle at 8hr intervals over 48 hours to obtain a total of 6 time point samples per clone. RNA from a total of 12 samples were extracted. Synthesis of target DNA was carried out as described in Bozdech, Z., S. Mok & A. P. Gupta, (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211 and used in replicate microarray hybridizations against a common RNA reference pool of 3D7 strain.

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Gene expression analysis of a double knockout of TCA cycle enzymes IDH and KDH

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