Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection.
Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.
Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USA.
Suresh M
SM
Ganesan
Joanne M
JM
Morrisey
Hangjun
H
Ke
Heather J
HJ
Painter
Kamal
K
Laroiya
Margaret A
MA
Phillips
Pradipsinh K
PK
Rathod
Michael W
MW
Mather
Akhil B
AB
Vaidya
D053626:Atovaquone / Q000494:pharmacology; D000090722:Dihydroorotate Dehydrogenase; D004351:Drug Resistance; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000295:instrumentation* / Q000379:methods
D000090722:Dihydroorotate Dehydrogenase; D029701:Saccharomyces cerevisiae Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; D053626:Atovaquone
Nature. 2008 Oct 9;455(7214):757-63
18843361
Antimicrob Agents Chemother. 1979 Dec;16(6):710-8
394674
Nature. 1997 Jan 23;385(6614):336-40
9002517
Mol Biochem Parasitol. 2006 Nov;150(1):118-21
16901558
Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10931-6
9380737
J Med Chem. 2009 Apr 9;52(7):1864-72
19296651
Int J Parasitol. 2002 Jan;32(1):1-14
11796117
Nature. 2002 Oct 3;419(6906):498-511
12368864
Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8716-20
10411941
Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1063-8
17213308
Nature. 2007 Mar 1;446(7131):88-91
17330044
Mol Biochem Parasitol. 2001 May;114(2):183-95
11378198
Nature. 2008 Oct 9;455(7214):799-803
18843368
Nature. 2002 Oct 3;419(6906):512-9
12368865
J Med Chem. 2008 Jun 26;51(12):3649-53
18522386
Biochim Biophys Acta. 1979 Feb 1;582(3):390-401
217438