Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10−6 structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0–9.7×10−9 mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations.
Vyšlo v časopise: Mitotic Evolution of Shows a Stable Core Genome but Recombination in Antigen Families. PLoS Genet 9(2): e32767. doi:10.1371/journal.pgen.1003293 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003293
1. WHO (2011) World Malaria Report 2011. http://www.who.int/malaria/world_malaria_report_2010/en/index.html.
2. O'BrienC, HenrichPP, PassiN, FidockDA (2011) Recent clinical and molecular insights into emerging artemisinin resistance in Plasmodium falciparum. Curr Opin Infect Dis 24 : 570–577.
3. A Phase 3 Trial of RTS,S/AS01 Malaria Vaccine in African Infants. N Engl J Med
4. GentonB, BetuelaI, FelgerI, Al-YamanF, AndersRF, et al. (2002) A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1–2b trial in Papua New Guinea. The Journal of Infectious Diseases 185 : 820–827.
5. Freitas-JuniorLH, BottiusE, PirritLA, DeitschKW, ScheidigC, et al. (2000) Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum. Nature 407 : 1018–1022.
6. KerrPJ, Ranford-CartwrightLC, WallikerD (1994) Proof of intragenic recombination in Plasmodium falciparum. Mol Biochem Parasitol 66 : 241–248.
7. DuffyMF, ByrneTJ, CarretC, IvensA, BrownGV (2009) Ectopic recombination of a malaria var gene during mitosis associated with an altered var switch rate. Journal of molecular biology 389 : 453–469.
8. JulianoJJ, TaylorSM, MeshnickSR (2009) Polymerase chain reaction adjustment in antimalarial trials: molecular malarkey? J Infect Dis 200 : 5–7.
9. JulianoJJ, PorterK, MwapasaV, SemR, RogersWO, et al. (2010) Exposing malaria in-host diversity and estimating population diversity by capture-recapture using massively parallel pyrosequencing. Proc Natl Acad Sci U S A 107 : 20138–20143.
10. RobinsonT, CampinoSG, AuburnS, AssefaSA, PolleySD, et al. (2011) Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients. PLoS ONE 6: e23204 doi:10.1371/journal.pone.0023204.
11. Paget-McNicolS, SaulA (2001) Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum. Parasitology 122 : 497–505.
12. SrivastavaIK, MorriseyJM, DarrouzetE, DaldalF, VaidyaAB (1999) Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites. Mol Microbiol 33 : 704–711.
13. LooareesuwanS, ViravanC, WebsterHK, KyleDE, HutchinsonDB, et al. (1996) Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg 54 : 62–66.
14. DhariaNV, SidhuAB, CasseraMB, WestenbergerSJ, BoppSE, et al. (2009) Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum. Genome Biology 10: R21.
15. GardnerM, HallN, FungE, WhiteO, BerrimanM, et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419 : 498–511.
16. MuJ, FerdigMT, FengX, JoyDA, DuanJ, et al. (2003) Multiple transporters associated with malaria parasite responses to chloroquine and quinine. Mol Microbiol 49 : 977–989.
17. RajDK, MuJ, JiangH, KabatJ, SinghS, et al. (2009) Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione. J Biol Chem 284 : 7687–7696.
18. NamTG, McNamaraCW, BoppS, DhariaNV, MeisterS, et al. (2011) A Chemical Genomic Analysis of Decoquinate, a Plasmodium falciparum Cytochrome b Inhibitor. ACS chemical biology 6 : 1214–1222.
19. FosterPL (2006) Methods for determining spontaneous mutation rates. Methods Enzymol 409 : 195–213.
20. NiccumBA, PoteauR, HammanGE, VaradaJC, DshalalowJH, et al. (2012) On an unbiased and consistent estimator for mutation rates. J Theor Biol 300 : 360–367.
21. LynchM, SungW, MorrisK, CoffeyN, LandryCR, et al. (2008) A genome-wide view of the spectrum of spontaneous mutations in yeast. Proceedings of the National Academy of Sciences of the United States of America 105 : 9272–9277.
22. Haag-LiautardC, DorrisM, MasideX, MacaskillS, HalliganDL, et al. (2007) Direct estimation of per nucleotide and genomic deleterious mutation rates in Drosophila. Nature 445 : 82–85.
23. NachmanMW, CrowellSL (2000) Estimate of the mutation rate per nucleotide in humans. Genetics 156 : 297–304.
24. RoachJC, GlusmanG, SmitAF, HuffCD, HubleyR, et al. (2010) Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science 328 : 636–639.
25. PologeLG, RavetchJV (1988) Large deletions result from breakage and healing of P. falciparum chromosomes. Cell 55 : 869–874.
26. BiggsBA, KempDJ, BrownGV (1989) Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro. Proc Natl Acad Sci U S A 86 : 2428–2432.
27. PatarapotikulJ, LangsleyG (1988) Chromosome size polymorphism in Plasmodium falciparum can involve deletions of the subtelomeric pPFrep20 sequence. Nucleic Acids Res 16 : 4331–4340.
28. ShirleyMW, BiggsBA, ForsythKP, BrownHJ, ThompsonJK, et al. (1990) Chromosome 9 from independent clones and isolates of Plasmodium falciparum undergoes subtelomeric deletions with similar breakpoints in vitro. Molecular and Biochemical Parasitology 40 : 137.
29. DhariaNV, PlouffeD, BoppSE, Gonzalez-PaezGE, LucasC, et al. (2010) Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites. Genome Res 20 : 1534–1544.
30. RottmannM, McNamaraC, YeungBK, LeeMC, ZouB, et al. (2010) Spiroindolones, a potent compound class for the treatment of malaria. Science 329 : 1175–1180.
31. CowmanA, GalatisD, ThompsonJ (1994) Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine. Proc Natl Acad Sci USA 91 : 1143–1147.
32. SidhuA, UhlemannA-C, ValderramosS, ValderramosJ-C, KrishnaS, et al. (2006) Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis 194 : 528–535.
33. FrankM, KirkmanL, CostantiniD, SanyalS, LavazecC, et al. (2008) Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum. Int J Parasitol 38 : 1099–1109.
34. FigueiredoLM, Freitas-JuniorLH, BottiusE, Olivo-MarinJC, ScherfA (2002) A central role for Plasmodium falciparum subtelomeric regions in spatial positioning and telomere length regulation. EMBO J 21 : 815.
35. ScherfA, MatteiD (1992) Cloning and characterization of chromosome breakpoints of Plasmodium falciparum: breakage and new telomere formation occurs frequently and randomly in subtelomeric genes. Nucleic Acids Res 20 : 1491–1496.
36. RathodPK, McErleanT, LeePC (1997) Variations in frequencies of drug resistance in Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 94 : 9389–9393.
37. LavstsenT, SalantiA, JensenAT, ArnotDE, TheanderTG (2003) Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions. Malar J 2 : 27.
38. BaruchDI, PasloskeBL, SinghHB, BiX, MaXC, et al. (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82 : 77.
39. McVeyM, LeeSE (2008) MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet 24 : 529–538.
40. ScherfA, Hernandez-RivasR, BuffetP, BottiusE, BenatarC, et al. (1998) Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum. EMBO J 17 : 5418.
41. ChenQ, FernandezV, SundstromA, SchlichtherleM, DattaS, et al. (1998) Developmental selection of var gene expression in Plasmodium falciparum. Nature 394 : 392–395.
42. RobertsDJ, CraigAG, BerendtAR, PinchesR, NashG, et al. (1992) Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357 : 689.
43. SmithJD, ChitnisCE, CraigAG, RobertsDJ, Hudson-TaylorDE, et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82 : 101.
44. Mancio-SilvaL, Rojas-MezaAP, VargasM, ScherfA, Hernandez-RivasR (2008) Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum. Journal of cell science 121 : 2046–2053.
45. Freitas-JuniorLH, Hernandez-RivasR, RalphSA, Montiel-CondadoD, Ruvalcaba-SalazarOK, et al. (2005) Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121 : 25.
46. PaysE, LaurentM, DelinteK, Van MeirvenneN, SteinertM (1983) Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei. Nucleic acids research 11 : 8137–8147.
47. VolkmanSK, SabetiPC, DeCaprioD, NeafseyDE, SchaffnerSF, et al. (2007) A genome-wide map of diversity in Plasmodium falciparum. Nat Genet 39 : 113–119.
48. World, Health, Organization (2008) Methods and techniques for clinical trials on antimalarial drug efficacy: Genotyping to Identify Parasite Populations. World Health Organization, Geneva.
49. WallikerD, QuakyiIA, WellemsTE, McCutchanTF, SzarfmanA, et al. (1987) Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 236 : 1661–1666.
50. RosarioV (1981) Cloning of naturally occurring mixed infections of malaria parasites. Science 212 : 1037–1038.
51. GardnerMJ, HallN, FungE, WhiteO, BerrimanM, et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419 : 498.
52. TragerW, JensonJ (1978) Cultivation of malarial parasites. Nature 273 : 621–622.
53. BeckHP (2002) Extraction and purification of Plasmodium parasite DNA. Methods Mol Med 72 : 159–163.
54. PlouffeD, BrinkerA, McNamaraC, HensonK, KatoN, et al. (2008) In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc Natl Acad Sci U S A 105 : 9059–9064.
55. OyolaSO, OttoTD, GuY, MaslenG, ManskeM, et al. (2012) Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes. BMC Genomics 13 : 1.
56. LiH, DurbinR (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 : 1754–1760.
57. LiH, HandsakerB, WysokerA, FennellT, RuanJ, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25 : 2078–2079.
58. DePristoMA, BanksE, PoplinR, GarimellaKV, MaguireJR, et al. (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43 : 491–498.
59. McKennaA, HannaM, BanksE, SivachenkoA, CibulskisK, et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20 : 1297–1303.
60. YangZ, NielsenR (2000) Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. Mol Biol Evol 17 : 32–43.
61. NishantKT, SinghND, AlaniE (2009) Genomic mutation rates: what high-throughput methods can tell us. Bioessays 31 : 912–920.
62. RoscheWA, FosterPL (2000) Determining mutation rates in bacterial populations. Methods 20 : 4–17.
63. Hernandez-RivasR, ScherfA (1997) Separation and mapping of chromosomes of parasitic protozoa. Mem Inst Oswaldo Cruz 92 : 815–819.
64. HinterbergK, ScherfA (1994) PFGE: improved conditions for rapid and high-resolution separation of Plasmodium falciparum chromosomes. Parasitol Today 10 : 225.
65. MaddisonWPaDRM (2011) Mesquite: a modular system for evolutionary analysis. Version 2.75 ed
66. LarkinMA, BlackshieldsG, BrownNP, ChennaR, McGettiganPA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23 : 2947–2948.
67. IstvanES, DhariaNV, BoppSE, GluzmanI, WinzelerEA, et al. (2011) Validation of isoleucine utilization targets in Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 108 : 1627–1632.
68. NkrumahLJ, MuhleRA, MouraPA, GhoshP, HatfullGF, et al. (2006) Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nat Methods 3 : 615–621.
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