piggyBac is an effective tool for functional analysis of the Plasmodium falciparumgenome

BMC Microbiology - 2009
Bharath Balu1, Chitra Chauhan1, Steven P. Maher1, Douglas A. Shoue2, Jessica C. Kissinger3, Malcolm J. Fraser2, John Adams1
1Department of Global Health, University of South Florida, 3720 Spectrum Blvd Suite 304, Tampa, Florida, 33612, USA
2Department of Biological Sciences University of Notre Dame Notre Dame, Indiana 46556 USA
3Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, 30602, USA

Tóm tắt

Abstract Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. Results In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development. Conclusion Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

Từ khóa


Tài liệu tham khảo

Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI: The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005, 434 (7030): 214-217. 10.1038/nature03342.

Yamey G: Roll Back Malaria: a failing global health campaign. Bmj. 2004, 328 (7448): 1086-1087. 10.1136/bmj.328.7448.1086.

Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ, et al: Discovery of gene function by expression profiling of the malaria parasite life cycle. Science. 2003, 301 (5639): 1503-1508. 10.1126/science.1087025.

Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The Transcriptome of the Intraerythrocytic Developmental Cycle of Plasmodium falciparum. PLoS Biol. 2003, 1 (1): 5-10.1371/journal.pbio.0000005.

Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, et al: A proteomic view of the Plasmodium falciparum life cycle. Nature. 2002, 419 (6906): 520-526. 10.1038/nature01107.

Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, Eling WM, Hall N, Waters AP, Stunnenberg HG, et al: Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature. 2002, 419 (6906): 537-542. 10.1038/nature01111.

LaCount DJ, Vignali M, Chettier R, Phansalkar A, Bell R, Hesselberth JR, Schoenfeld LW, Ota I, Sahasrabudhe S, Kurschner C, et al: A protein interaction network of the malaria parasite Plasmodium falciparum. Nature. 2005, 438 (7064): 103-107. 10.1038/nature04104.

Date SV, Stoeckert CJ: Computational modeling of the Plasmodium falciparum interactome reveals protein function on a genome-wide scale. Genome Res. 2006, 16 (4): 542-549. 10.1101/gr.4573206.

O'Donnell RA, Freitas-Junior LH, Preiser PR, Williamson DH, Duraisingh M, McElwain TF, Scherf A, Cowman AF, Crabb BS: A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes. Embo J. 2002, 21 (5): 1231-1239. 10.1093/emboj/21.5.1231.

Greenbaum DC: Is chemical genetics the new frontier for malaria biology?. Trends Pharmacol Sci. 2008, 29 (2): 51-56. 10.1016/j.tips.2007.11.008.

Carlson CM, Frandsen JL, Kirchhof N, McIvor RS, Largaespada DA: Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse. Proc Natl Acad Sci USA. 2005, 102 (47): 17059-17064. 10.1073/pnas.0502974102.

St Johnston D: The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet. 2002, 3 (3): 176-188. 10.1038/nrg751.

Grimm S: The art and design of genetic screens: mammalian culture cells. Nat Rev Genet. 2004, 5 (3): 179-189. 10.1038/nrg1291.

Hayes F: Transposon-based strategies for microbial functional genomics and proteomics. Annu Rev Genet. 2003, 37: 3-29. 10.1146/annurev.genet.37.110801.142807.

Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, Fraser MJ: Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology. 1989, 172 (1): 156-169. 10.1016/0042-6822(89)90117-7.

Fraser MJ, Brusca JS, Smith GE, Summers MD: Transposon-mediated mutagenesis of a baculovirus. Virology. 1985, 145 (2): 356-361. 10.1016/0042-6822(85)90172-2.

Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T: Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell. 2005, 122 (3): 473-483. 10.1016/j.cell.2005.07.013.

Lobo NF, Fraser TS, Adams JA, Fraser MJ: Interplasmid transposition demonstrates piggyBac mobility in vertebrate species. Genetica. 2006, 128 (1–3): 347-357. 10.1007/s10709-006-7165-2.

Morales ME, Mann VH, Kines KJ, Gobert GN, Fraser MJ, Kalinna BH, Correnti JM, Pearce EJ, Brindley PJ: piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni. Faseb J. 2007, 21 (13): 3479-3489. 10.1096/fj.07-8726com.

Thibault ST, Singer MA, Miyazaki WY, Milash B, Dompe NA, Singh CM, Buchholz R, Demsky M, Fawcett R, Francis-Lang HL, et al: A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet. 2004, 36 (3): 283-287. 10.1038/ng1314.

Balu B, Shoue DA, Fraser MJ, Adams JH: High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac. Proc Natl Acad Sci USA. 2005, 102 (45): 16391-16396. 10.1073/pnas.0504679102.

Crabb BS, Triglia T, Waterkeyn JG, Cowman AF: Stable transgene expression in Plasmodium falciparum. Molecular and Biochemical Parasitology. 1997, 90: 131-144. 10.1016/S0166-6851(97)00143-6.

Kissinger JC, Brunk BP, Crabtree J, Fraunholz MJ, Gajria B, Milgram AJ, Pearson DS, Schug J, Bahl A, Diskin SJ, et al: The Plasmodium genome database. Nature. 2002, 419 (6906): 490-492. 10.1038/419490a.

Li X, Harrell RA, Handler AM, Beam T, Hennessy K, Fraser MJ: piggyBac internal sequences are necessary for efficient transformation of target genomes. Insect Mol Biol. 2005, 14 (1): 17-30. 10.1111/j.1365-2583.2004.00525.x.

Persson KE, Lee CT, Marsh K, Beeson JG: Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies. J Clin Microbiol. 2006, 44 (5): 1665-1673. 10.1128/JCM.44.5.1665-1673.2006.

Liu J, Gluzman IY, Drew ME, Goldberg DE: The role of Plasmodium falciparum food vacuole plasmepsins. J Biol Chem. 2005, 280 (2): 1432-1437. 10.1074/jbc.M409740200.

Ryder E, Russell S: Transposable elements as tools for genomics and genetics in Drosophila. Brief Funct Genomic Proteomic. 2003, 2 (1): 57-71. 10.1093/bfgp/2.1.57.

Lobo NF, Hua-Van A, Li X, Nolen BM, Fraser MJ: Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vector. Insect Mol Biol. 2002, 11 (2): 133-139. 10.1046/j.1365-2583.2002.00317.x.

Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, et al: Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol. 2000, 18 (1): 81-84. 10.1038/71978.

Grossman GL, Rafferty CS, Fraser MJ, Benedict MQ: The piggyBac element is capable of precise excision and transposition in cells and embryos of the mosquito, Anopheles gambiae. Insect Biochem Mol Biol. 2000, 30 (10): 909-914. 10.1016/S0965-1748(00)00092-8.

Balu B, Adams JH: Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis. Cell Microbiol. 2006, 8 (10): 1529-1536. 10.1111/j.1462-5822.2006.00776.x.

Maier AG, Rug M, O'Neill MT, Brown M, Chakravorty S, Szestak T, Chesson J, Wu Y, Hughes K, Coppel RL, et al: Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell. 2008, 134 (1): 48-61. 10.1016/j.cell.2008.04.051.

Coulson RM, Hall N, Ouzounis CA: Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum. Genome Res. 2004, 14 (8): 1548-1554. 10.1101/gr.2218604.

Collart MA: Global control of gene expression in yeast by the Ccr4-Not complex. Gene. 2003, 313: 1-16. 10.1016/S0378-1119(03)00672-3.

Shock JL, Fischer KF, DeRisi JL: 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 (7): R134-10.1186/gb-2007-8-7-r134.

Aravind L, Iyer LM, Wellems TE, Miller LH: Plasmodium biology: genomic gleanings. Cell. 2003, 115 (7): 771-785. 10.1016/S0092-8674(03)01023-7.

Luan S: Protein phosphatases in plants. Annu Rev Plant Biol. 2003, 54: 63-92. 10.1146/annurev.arplant.54.031902.134743.

Saito H, Tatebayashi K: Regulation of the osmoregulatory HOG MAPK cascade in yeast. J Biochem. 2004, 136 (3): 267-272. 10.1093/jb/mvh135.

Heideker J, Lis ET, Romesberg FE: Phosphatases, DNA Damage Checkpoints and Checkpoint Deactivation. Cell Cycle. 2007, 6 (24): 3058-3064.

Delorme V, Cayla X, Faure G, Garcia A, Tardieux I: Actin dynamics is controlled by a casein kinase II and phosphatase 2C interplay on Toxoplasma gondii Toxofilin. Mol Biol Cell. 2003, 14 (5): 1900-1912. 10.1091/mbc.E02-08-0462.

Gilbert LA, Ravindran S, Turetzky JM, Boothroyd JC, Bradley PJ: Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryot Cell. 2007, 6 (1): 73-83. 10.1128/EC.00309-06.

Bhattacharjee S, van Ooij C, Balu B, Adams JH, Haldar K: Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte. Blood. 2008, 111 (4): 2418-2426. 10.1182/blood-2007-09-115279.

Fidock DA, Wellems TE: Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. Proc Natl Acad Sci USA. 1997, 94 (20): 10931-10936. 10.1073/pnas.94.20.10931.

Wickham ME, Rug M, Ralph SA, Klonis N, McFadden GI, Tilley L, Cowman AF: Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes. Embo J. 2001, 20 (20): 5636-5649. 10.1093/emboj/20.20.5636.

Mamoun CB, Gluzman IY, Goyard S, Beverley SM, Goldberg DE: A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum. Proc Natl Acad Sci USA. 1999, 96 (15): 8716-8720. 10.1073/pnas.96.15.8716.

Kadekoppala M, Kline K, Akompong T, Haldar K: Stable expression of a new chimeric fluorescent reporter in the human malaria parasite Plasmodium falciparum. Infect Immun. 2000, 68 (4): 2328-2332. 10.1128/IAI.68.4.2328-2332.2000.

Li Q, Gerena L, Xie L, Zhang J, Kyle D, Milhous W: Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum v itally stained with YOYO-1. Cytometry A. 2007, 71 (5): 297-307.

Myrick A, Munasinghe A, Patankar S, Wirth DF: Mapping of the Plasmodium falciparum multidrug resistance gene 5'-upstream region, and evidence of induction of transcript levels by antimalarial drugs in chloroquine sensitive parasites. Mol Microbiol. 2003, 49 (3): 671-683. 10.1046/j.1365-2958.2003.03597.x.

Golightly LM, Mbacham W, Daily J, Wirth DF: 3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum. Mol Biochem Parasitol. 2000, 105 (1): 61-70. 10.1016/S0166-6851(99)00165-6.

Thông tin
Thông tin xuất bản

BMC Microbiology

Thông tin tác giả