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Mutations in the Plasmodium falciparum chloroquine resistance transporter: Balancing drug resistance and fitness.

機譯:惡性瘧原蟲氯喹抗性轉運蛋白中的突變:平衡耐藥性和適應性。

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Mutations in the Plasmodium falciparum chioroquine resistance transporter (PfCRT) render malaria parasites resistant to chloroquine. High rates of resistance to chloroquine have prompted many malarious countries to switch to alternative therapies. In Malawi, the replacement of chloroquine has led to the reemergence of wild type chioroquine-sensitive parasites, a finding that was attributed to a decreased fitness of parasites with mutant pfcrt. However, in Southeast Asia mutant parasites were maintained in the population after the replacement of chioroquine with various other antimalarial drugs. I reasoned that local Southeast Asian pfcrt alleles might either be selected by the regionally used replacement drugs or do not confer a fitness cost as observed with chloroquine-resistant alleles in Malawi.;To examine the properties of Southeast Asian PfCRT haplotypes, I used an allelic exchange strategy to create isogenic parasites that expressed the Cam734 pfcrt allele from Cambodia and the Phi and Ph2 alleles from the Philippines, respectively. To test the influence of PfCRT haplotype on drug susceptibility, I assayed these recombinant lines with various antimalarial drugs. I found that not only the established K76T mutation but also the C725 polymorphism influences chloroquine resistance in the Philippine haplotypes. The only amino acid difference between the two Philippine PfCRT haplotypes occurs in residue 72 and leads to chloroquine sensitivity in Phi (C72) and chloroquine resistance in Ph2 (72S). Additionally, different levels of amodiaquine resistance were found to depend on the geographic origin of the mutant pfcrt allele, with high resistance in the Philippines haplotypes, intermediate levels in Cambodian and Brazilian haplotypes and low levels in the Asian/African haplotype. I also showed that lumefantrine susceptibilities are influenced by pfcrt alleles, with the wild type allele conferring resistance and mutant alleles leading to susceptibility to this drug. Examination of recombinant parasites, which expressed either PfCRT K76 or 76T, revealed additionally that this polymorphic residue was the main determinant of pfcrt-dependent lumefantrine susceptibility and established the validity of K76T as a marker for lumefantrine resistance. These in vitro data has been confirmed by a clinical trial in Tanzania, which investigated the association of lumefantrine treatment with the recrudescence of parasites expressing wild type pfcrt alleles. The results obtained from these drug susceptibility experiments provide important information for the treatment of falciparum malaria with the appropriate drug in Cambodia and the Philippines depending on the predominant pfcrt allele on a regional level.;I also utilized the recombinant lines I generated to test whether PfCRT haplotype influences parasite fitness, as has been suggested from observations in Malawi. For this, a fitness assay was designed that utilized pyrosequencing to quantify changes in the abundance of two co-cultured isogenic parasite lines over time. Indeed, expression of different pfcrt alleles led to distinct relative fitness values. Interestingly, the recombinant line expressing Ph1pfcrt was more fit but less resistant to chloroquine than the Ph2 expressing recombinant and vice versa, which could be attributed to the exchange of only one amino acid at position 72. Also, fitness results from the Cam734 allele from Cambodia were intriguing because parasites expressing Cam734 pfcrt displayed an even higher relative fitness value than parasites expressing the wild type allele despite increased chloroquine resistance. This has important implications for the future geographical distribution of this mutant Cambodian PfCRT haplotype, possibly expanding at the expense of the wild type allele and other less fit mutant parasites due to its high fitness.;Finally, I created a parasite line that stably expresses PfCRT fused to a tandem affinity purification tag, which is delivered to the digestive vacuole (DV) surface. Besides the essential hemoglobin degradation pathway, few other metabolic processes have been localized to the DV. The expression of tagged PfCRT on the DV surface allows the immuno-precipitation of highly purified DV preparations as well as PfCRT binding partners, which then can be subjected to mass spectrometry analysis. This tool might lead to the identification of new drug targets and assign new metabolic pathways to the DV.
機譯:惡性瘧原蟲chioroquine抗性轉運蛋白(PfCRT)中的突變使瘧原蟲對氯喹具有抗性。對氯喹的高耐藥率促使許多病態(tài)的國家轉向替代療法。在馬拉維,替換氯喹導致野生型對氯喹敏感的寄生蟲重新出現(xiàn),這一發(fā)現(xiàn)歸因于突變型pfcrt降低了寄生蟲的適應性。然而,在東南亞,用各種其他抗瘧疾藥物代替氯喹后,突變體寄生蟲仍在種群中得以維持。我認為,東南亞的pfcrt等位基因可能是由當?shù)厥褂玫奶娲幬镞x擇的,或者不像馬拉維耐氯喹的等位基因那樣帶來健身費用。為了檢查東南亞PfCRT單倍型的特性,我使用了一個等位基因交換策略,以創(chuàng)建分別表達來自柬埔寨的Cam734 pfcrt等位基因和來自菲律賓的Phi和Ph2等位基因的等基因寄生蟲。為了測試PfCRT單倍型對藥物敏感性的影響,我用各種抗瘧藥分析了這些重組品系。我發(fā)現(xiàn),不僅已建立的K76T突變,而且C725多態(tài)性都會影響菲律賓單體型的氯喹抗性。兩種菲律賓PfCRT單倍型之間唯一的氨基酸差異出現(xiàn)在殘基72中,并導致Phi(C72)中的氯喹敏感性和Ph2(72S)中的氯喹抗性。此外,發(fā)現(xiàn)不同水平的氨二喹抗性取決于突變的pfcrt等位基因的地理起源,菲律賓單倍型具有高抗性,柬埔寨和巴西單倍型具有中等水平,而亞洲/非洲單倍型具有低水平。我還表明,lumfantrine敏感性受pfcrt等位基因影響,野生型等位基因賦予抗藥性,而突變等位基因則導致對該藥物的敏感性。對表達PfCRT K76或76T的重組寄生蟲進行的檢查另外顯示,該多態(tài)性殘基是pfcrt依賴的lumantantrine敏感性的主要決定因素,并確定了K76T作為lumantantrine抗性標記的有效性。這些體外數(shù)據(jù)已經(jīng)在坦桑尼亞的一項臨床試驗中得到了證實,該試驗研究了熒光素治療與表達野生型pfcrt等位基因的寄生蟲再發(fā)的關系。從這些藥物敏感性實驗獲得的結果為在柬埔寨和菲律賓使用適當藥物治療惡性瘧疾提供了重要信息,具體取決于區(qū)域一級的主要pfcrt等位基因。我還利用我產(chǎn)生的重組品系測試了PfCRT正如在馬拉維的觀察所表明的那樣,單體型會影響寄生蟲的適應能力。為此,設計了一種適應性測定法,該方法利用焦磷酸測序來量化兩個共培養(yǎng)的等基因寄生蟲系隨時間的豐度變化。實際上,不同pfcrt等位基因的表達導致了不同的相對適應度值。有趣的是,表達Ph1pfcrt的重組品系比表達Ph2的重組品更適合,但對氯喹的抵抗力較低,反之亦然,這可能歸因于72位僅交換一個氨基酸。此外,來自柬埔寨的Cam734等位基因也具有適應性之所以令人著迷,是因為盡管對氯喹的抵抗力增強,但表達Cam734 pfcrt的寄生蟲比表達野生型等位基因的寄生蟲顯示出更高的相對適應度值。這對于該突變型柬埔寨PfCRT單倍型的未來地理分布具有重要意義,可能由于其高適應性而以野生型等位基因和其他不太適合的突變型寄生蟲為代價進行擴展;最后,我創(chuàng)建了一個穩(wěn)定表達PfCRT的寄生蟲系。融合到串聯(lián)親和純化標簽上,然后將其傳遞到消化液泡(DV)表面。除了必需的血紅蛋白降解途徑外,很少有其他代謝過程局限于DV。標記的PfCRT在DV表面上的表達可實現(xiàn)高純度DV制劑以及PfCRT結合伴侶的免疫沉淀,然后可對其進行質譜分析。該工具可能導致新藥靶標的鑒定,并為DV分配新的代謝途徑。

著錄項

  • 作者

    Petersen, Ines.;

  • 作者單位

    Columbia University.;

  • 授予單位 Columbia University.;
  • 學科 Biology Microbiology.;Biology Parasitology.
  • 學位 Ph.D.
  • 年度 2010
  • 頁碼 137 p.
  • 總頁數(shù) 137
  • 原文格式 PDF
  • 正文語種 eng
  • 中圖分類
  • 關鍵詞

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