Welcome to Open Science
Contact Us
Home Books Journals Submission Open Science Join Us News
Chloroquine Tolerance in 3D7 Strain of P. falciparum was Associated with Change in Allelic Structure and not Pfcrt and Pfmdr1 Transporter Genes nor DNA Methylation
Current Issue
Volume 2, 2014
Issue 5 (October)
Pages: 100-107   |   Vol. 2, No. 5, October 2014   |   Follow on         
Paper in PDF Downloads: 19   Since Aug. 28, 2015 Views: 1440   Since Aug. 28, 2015
Martin Maina Wahome, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya; Institute of Tropical Medicine and Infectious Diseases, Jomo Kenyatta University of Agriculture & Technology, Nairobi, Kenya.
Josphat Nyandieka Nyataya, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
Hosea Miima Akala, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
George Odhiambo Awinda, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
Beth Kinya Mutai, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
Rebecca Waihenya, Department of Zoology, Jomo Kenyatta University of Agriculture & Technology, Nairobi, Kenya.
Edwin Kamau, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
Sabah Ahmed Omar, Centre of Geographical Medicine Research Coast, Kenya Medical Research Institute, Kilifi, Kenya.
John Njenga Waitumbi, Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
Development of anti-malarial resistance occurs at a fitness cost to the parasites. It makes sense to assume that P. falciparum would first use alternative mechanisms to survive transient drug pressure, and only resort to genetic fixation if the pressure is sustained. In this study, development of chloroquine (CQ) tolerance was assessed by DNA methylation, allelic diversity and genetic changes at Pfcrt and Pfmdr1. A CQ sensitive 3D7 strain of P. falciparum was cloned by limiting dilution and the derived population exposed to increasing CQ concentrations of 4.51 ng/mL, 5.99 ng/mL and 7.15 ng/mL corresponding to 10%, 30% and 50% inhibitory concentrations (IC) of the parental population. The surviving parasite density at each drug level was determined by SYBR Green I fluorassay. Allelic diversity of CQ unexposed and those surviving drug pressure were assessed by nested PCR that targeted the polymorphic regions of msp1, msp2 and glurp. Global DNA methylation at 5-methylCytosine (5-mC) was assessed by ELISA. Mutations at Pfcrt and Pfmdr1 genes were assessed by single nucleotide polymorphisms (SNPs). The CQ unexposed population had an IC50 of 7.03±1.37 ng/mL, one K1 allele (248 bp), two IC3D7 (482 bp and 596 bp) and one 800 bp glurp. Pfcrt and Pfmdr1 were wild type. 5-mC DNA methylation was not detectable. Post CQ exposure at 4.51 ng/mL and 5.99 ng/mL, IC50 increased to 10.5 ng/mL and 15.05 ng/mL respectively. Parasite growth at 7.15 ng/mL of CQ was minimal IC50 could not be determined. At 5.99 ng/mL of CQ, change in parasite structure was marked by allele reduction in parasites carrying the K1 and the 596 bp IC3D7 alleles. At 7.15 ng/mL of CQ, parasites with these two alleles were lost, but the 482 bp IC3D7 and 800 bp glurp clones survived. CQ tolerant populations remained wild type at Pfcrt and Pfmdr1. 5-mC DNA methylation was not observed in any of the derived parasite populations. These data suggest that, development of CQ tolerance starts by clonal selection. In absence of genetic or epigenetic changes to the surviving clones, further studies are needed to elucidate how CQ induced changes at Pfcrt and Pfmdr1 genes eventually occur.
Plasmodium falciparum, DNA Methylation, Pfcrt, Pfmdr1, Single Nucleotide Polymorphisms, Chloroquine Resistance, Genetic Diversity
WHO: World Malaria Report 2012. Geneva: World Health Organization; 2012.
Winstanley PA: Chemotherapy for falciparum malaria: the armoury, the problems and the prospects. Parasitology today (Personal ed 2000, 16(4):146-153.
Hastings IM, Watkins WM, White NJ: The evolution of drug-resistant malaria: the role of drug elimination half-life. Philosophical transactions of the Royal Society of London 2002, 357(1420):505-519.
Wernsdorfer WH: Epidemiology of drug resistance in malaria. Acta tropica 1994, 56(2-3):143-156.
Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR: Epidemiology of drug-resistant malaria. The Lancet infectious diseases 2002, 2(4):209-218.
Moore DV, Lanier JE: Observations on two Plasmodium falciparum infections with an abnormal response to chloroquine. The American journal of tropical medicine and hygiene 1961, 10:5-9.
Wernsdorfer WH, Payne D: The dynamics of drug resistance in Plasmodium falciparum. Pharmacology & therapeutics 1991, 50(1):95-121.
Campbell CC, Chin W, Collins WE, Teutsch SM, Moss DM: Chloroquine-resistant Plasmodium falciparum from East Africa: cultivation and drug sensitivity of the Tanzanian I/CDC strain from an American tourist. Lancet 1979, 2(8153):1151-1154.
Fogh S, Jepsen S, Effersoe P: Chloroquine-resistant Plasmodium falciparum malaria in Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 1979, 73(2):228-229.
Greenwood B: The use of anti-malarial drugs to prevent malaria in the population of malaria-endemic areas. The American journal of tropical medicine and hygiene 2004, 70(1):1-7.
Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF: Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 1990, 345(6272):255-258.
Wellems TE, Walker-Jonah A, Panton LJ: Genetic mapping of the chloroquine-resistance locus on Plasmodium falciparum chromosome 7. Proceedings of the National Academy of Sciences of the United States of America 1991, 88(8):3382-3386.
Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Coulibaly D, Dicko A, Su XZ, Nomura T et al: A molecular marker for chloroquine-resistant falciparum malaria. The New England journal of medicine 2001, 344(4):257-263.
Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW et al: Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Molecular cell 2000, 6(4):861-871.
Hayward R, Saliba KJ, Kirk K: pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum. Molecular microbiology 2005, 55(4):1285-1295.
Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF: Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 2000, 403(6772):906-909.
Merrick CJ, Duraisingh MT: Epigenetics in Plasmodium: what do we really know? Eukaryotic cell 2010, 9(8):1150-1158.
Jones PA, Baylin SB: The fundamental role of epigenetic events in cancer. Nature reviews 2002, 3(6):415-428.
Ralph SA, Scherf A: The epigenetic control of antigenic variation in Plasmodium falciparum. Current opinion in microbiology 2005, 8(4):434-440.
Hakimi MA, Deitsch KW: Epigenetics in Apicomplexa: control of gene expression during cell cycle progression, differentiation and antigenic variation. Current opinion in microbiology 2007, 10(4):357-362.
Basco LK, Le Bras J: Reversal of chloroquine resistance with desipramine in isolates of Plasmodium falciparum from Central and West Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 1990, 84(4):479-481.
van Schalkwyk DA, Walden JC, Smith PJ: Reversal of chloroquine resistance in Plasmodium falciparum using combinations of chemosensitizers. Antimicrobial agents and chemotherapy 2001, 45(11):3171-3174.
Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimde AA, Kouriba B, Taylor TE, Plowe CV: Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. The Journal of infectious diseases 2003, 187(12):1870-1875.
Mita T, Kaneko A, Lum JK, Bwijo B, Takechi M, Zungu IL, Tsukahara T, Tanabe K, Kobayakawa T, Bjorkman A: Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. The American journal of tropical medicine and hygiene 2003, 68(4):413-415.
Trager W, Jensen JB: Human malaria parasites in continuous culture. Science (New York, NY 1976, 193(4254):673-675.
Akala HM, Eyase FL, Cheruiyot AC, Omondi AA, Ogutu BR, Waters NC, Johnson JD, Polhemus ME, Schnabel DC, Walsh DS: Antimalarial drug sensitivity profile of western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay and molecular analysis. The American journal of tropical medicine and hygiene 2011, 85(1):34-41.
Amor A, Toro C, Fernandez-Martinez A, Baquero M, Benito A, Berzosa P: Molecular markers in plasmodium falciparum linked to resistance to anti-malarial drugs in samples imported from Africa over an eight-year period (2002-2010): impact of the introduction of artemisinin combination therapy. Malaria journal 2012, 11:100. Protocol available online at http://medschool.umaryland.edu/cvd/plowe.html.
Alam MT, de Souza DK, Vinayak S, Griffing SM, Poe AC, Duah NO, Ghansah A, Asamoa K, Slutsker L, Wilson MD et al: Selective sweeps and genetic lineages of Plasmodium falciparum drug -resistant alleles in Ghana. The Journal of infectious diseases 2011, 203(2):220-227.
Kamau E, Alemayehu S, Feghali KC, Tolbert LS, Ogutu B, Ockenhouse CF: Development of a TaqMan Allelic Discrimination assay for detection of single nucleotides polymorphisms associated with anti-malarial drug resistance. Malaria journal 2012, 11:23.
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif 2001, 25(4):402-408.
Liljander A, Wiklund L, Falk N, Kweku M, Martensson A, Felger I, Farnert A: Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2). Malaria journal 2009, 8:78.
Buckling A, Crooks L, Read A: Plasmodium chabaudi: effect of antimalarial drugs on gametocytogenesis. Experimental parasitology 1999, 93(1):45-54.
Buckling A, Ranford-Cartwright LC, Miles A, Read AF: Chloroquine increases Plasmodium falciparum gametocytogenesis in vitro. Parasitology 1999, 118 ( Pt 4):339-346.
Cooper RA, Ferdig MT, Su XZ, Ursos LM, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE: Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum. Molecular pharmacology 2002, 61(1):35-42.
Eyase FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, Juma D, Yeda R, Andagalu B, Wanja E et al: The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P. falciparum samples during 2008-2011. PloS one 2013, 8(5):e64299.
Eyase FL, Akala HM, Johnson JD, Walsh DS: Inhibitory activity of ferroquine, versus chloroquine, against western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay. The American journal of tropical medicine and hygiene 2011, 85(6):984-988.
Mbaisi A, Liyala P, Eyase F, Achilla R, Akala H, Wangui J, Mwangi J, Osuna F, Alam U, Smoak BL et al: Drug susceptibility and genetic evaluation of Plasmodium falciparum isolates obtained in four distinct geographical regions of Kenya. Antimicrobial agents and chemotherapy 2004, 48(9):3598-3601.
Peel SA, Merritt SC, Handy J, Baric RS: Derivation of highly mefloquine-resistant lines from Plasmodium falciparum in vitro. The American journal of tropical medicine and hygiene 1993, 48(3):385-397.
Gosi P, Lanteri CA, Tyner SD, Se Y, Lon C, Spring M, Char M, Sea D, Sriwichai S, Surasri S et al: Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia. Malaria journal 2013, 12:403.
Lim AS, Cowman AF: Plasmodium falciparum: chloroquine selection of a cloned line and DNA rearrangements. Experimental parasitology 1996, 83(3):283-294.
IE AE, ElGhazali G, TM AE, Hamad AA, Babiker HA, Elbashir MI, Giha HA: Allelic polymorphism of MSP2 gene in severe P. falciparum malaria in an area of low and seasonal transmission. Parasitology research 2007, 102(1):29-34.
Beale GH: The genetics of drug resistance in malaria parasites. Bulletin of the World Health Organization 1980, 58(5):799-804.
Gomez-Diaz E, Jorda M, Peinado MA, Rivero A: Epigenetics of host-pathogen interactions: the road ahead and the road behind. PLoS pathogens 2012, 8(11):e1003007.
Pollack Y, Kogan N, Golenser J: Plasmodium falciparum: evidence for a DNA methylation pattern. Experimental parasitology 1991, 72(4):339-344.
Ponts N, Fu L, Harris EY, Zhang J, Chung DW, Cervantes MC, Prudhomme J, Atanasova-Penichon V, Zehraoui E, Bunnik EM et al: Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum. Cell host & microbe 2013, 14(6):696-706.
Choi SW, Keyes MK, Horrocks P: LC/ESI-MS demonstrates the absence of 5-methyl-2'-deoxycytosine in Plasmodium falciparum genomic DNA. Molecular and biochemical parasitology 2006, 150(2):350-352.
Coulson RM, Hall N, Ouzounis CA: Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum. Genome research 2004, 14(8):1548-1554.
Open Science Scholarly Journals
Open Science is a peer-reviewed platform, the journals of which cover a wide range of academic disciplines and serve the world's research and scholarly communities. Upon acceptance, Open Science Journals will be immediately and permanently free for everyone to read and download.
Office Address:
228 Park Ave., S#45956, New York, NY 10003
Phone: +(001)(347)535 0661
Copyright © 2013-, Open Science Publishers - All Rights Reserved