Investigating transcriptional regulation of Plasmodium falciparum upon drug perturbation
© Brummlen et al; licensee BioMed Central Ltd. 2010
Published: 20 October 2010
Gene regulation of the malaria parasite Plasmodium falciparum has proven to be complex with evidence supporting both transcriptional [1, 2] and post-transcriptional level control [3, 4]. Transcriptional profiling of environmentally perturbed malaria parasites can reveal functionally related genes with common regulatory mechanisms that are responsive to external stimuli . By interrogating microarray data derived from P. falciparum populations treated with several drug classes, a subset of four genes potentially involved with the parasite's survival mechanisms following drug perturbation were identified, i.e. general stress response genes. In addition, data from two independent functional genomics investigations on the response of P. falciparum parasites to polyamine biosynthesis inhibitors (DFMO/MDL73811 co-inhibition  and cyclohexylamine ), revealed four genes of which the transcript abundance appear to be uniquely affected upon polyamine depletion only [5, 6], i.e. perturbation-specific stress response genes. The regulation of this generalized versus perturbation-specific transcriptional responses was subsequently investigated.
In order to investigate transcriptional regulation of P. falciparum, stable transfection methodology was established through the use of green fluorescent protein and luciferase as transgene expression markers . Subsequently, the regions 1500 bp upstream of the coding sequence (presumably containing the promoters) of the eight genes were inserted into the transfection vector driving luciferase expression, with the heat shock protein 86 promoter as control. Parasite transfection was performed and transfectants were selected for resistance against the antifolate WR99210.
Stable lines of the P. falciparum transfectants were exposed to a variety of compounds from different antimalarial drug classes including two polyamine biosynthesis inhibitors (i.e. artemisinin, mefloquine,doxycycline, DFMO and cyclohexylamine) and luciferase activity was quantified as a measure of promoter activity. The altered luciferase activity was used to determine whether the observed transcriptional response upon drug perturbation were indeed the result of true transcriptional control at the promoter level as opposed to alteration of mRNA stability or other post-transcriptional means.
- De Silva E, Gehrke A, Olszewski K, León I, Chahal J, Bulyk M, Llinás M: Specific DNA-binding by Apicomplexan AP2 transcription factors. Proc Natl Acad Sci USA. 2008, 105: 8393-8. 10.1073/pnas.0801993105.PubMed CentralView ArticlePubMedGoogle Scholar
- Hu G, Cabrera A, Kono M, Mok S, Chaal B, Haase S, Engelberg K, Cheemadan S, Spielmann T, Preiser P: Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum. Nat Biotechnol. 2010, 28: 91-8. 10.1038/nbt.1597.View ArticlePubMedGoogle Scholar
- Mair G, Braks J, Garver L, Dimopoulos G, Hall N, Wiegant J, Dirks R, Khan S, Janse C, Waters A: Translational repression is essential for Plasmodium sexual development and mediated by DDX6-type RNA helicase. Science. 2006, 313: 667-9. 10.1126/science.1125129.PubMed CentralView ArticlePubMedGoogle Scholar
- Shock J, Fischer K, DeRisi J: Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle. Genome Biol. 2007, 8: R134-10.1186/gb-2007-8-7-r134.PubMed CentralView ArticlePubMedGoogle Scholar
- van Brummelen AC, Olszewski KL, Wilinski D, Llinas M, Louw AI, Birkholtz L-M: Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses. J Biol Chem. 2009, 284: 4635-46. 10.1074/jbc.M807085200.PubMed CentralView ArticlePubMedGoogle Scholar
- Becker J, Mtwisha L, Crampton B, Stoychev S, Van Brummelen A, Reeksting S, Louw A, Birkholtz L-M, Mancama D: Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels. BMC Genomics. 2010, 11: 235-10.1186/1471-2164-11-235.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Brummelen AC, Becker JVW: Establishing malaria parasite transfection technology in South Africa. In 22nd South African Society for Biochemistry and Molecular Biology Congress. 2010, South Africa: Bloemfontein, South AfricaGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.