The pH of the digestive vacuole of<i>Plasmodium falciparum</i>is not associated with chloroquine resistance

Hayward, Rhys; Saliba, Kevin J.; Kirk, Kiaran

doi:10.1242/jcs.02795

Cited by 125 publications

(152 citation statements)

References 79 publications

(143 reference statements)

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“…Hydropathy values of amino acid side chains within a standardized synthetic polypeptide structure were determined at pH 5.0 by Kovacs et al (39). This pH value is within the measured pH range for the P. falciparum DV lumen (5,28), where the PfCRT position 76 amino acid is predicted to face (Fig. 2) (43).…”

Section: I-369fsupporting

confidence: 81%

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

Griffin

Hoke

Samarakoon

et al. 2012

Antimicrob Agents Chemother

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eThe Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (؊)-quinine and (؉)-quinidine. The potency of (؉)-isomers is greater than the (؊)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC 50 ratio of (؊)/(؉) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (؊) and (؉) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.A lkaloids from the Cinchona tree, exemplified by quinine (QN) and quinidine (QD), have proven to be an important source of antimalarial therapies, especially after resistance to chloroquine (CQ) emerged. QN remains a first-line drug in the treatment of severe malaria in many parts of the world, even with increased use of artemisinin-based combination therapies (70). The Cinchona alkaloids (CA) are aryl amino alcohols where the aryl group is a quinoline (quinoline aminoalcohols). They have four chiral centers, two of which, C-8 and C-9, can have different configurations (66) (Fig. 1). Among the pharmacologically active 8,9-erythro isomers, QN, hydroquinine (HQN), and cinchonidine (CD) all present the S configuration around C-8 and the R configuration at C-9 and are levorotatory (rotate plane polarized light in an anticlockwise [Ϫ] direction). The reverse ordering, R at C-8 and S at C-9, occurs in the respective dextrorotatory (rotate plane polarized light in a clockwise [ϩ] direction) diastereomers, QD, hydroquinidine (HQD), and cinchonine (CN). The 8,9-threo diastereomers 9-epiquinine (EQN) and 9-epiquinidine (EQD) are 8S, 9S, and 8R, 9R, respectively.Against Plasmodium falciparum, the CA diastereomer pairs have a well-established in ...

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Section: I-369fsupporting

confidence: 81%

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

Griffin

Hoke

Samarakoon

et al. 2012

Antimicrob Agents Chemother

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“…A separate study however, reported a slight alkalinization of CQR parasite DVs compared with CQS parasites, although this difference disappeared at lower pHsensitive dye concentrations [33]. These discrepancies in reported DV pH may be more reflective of differences in measurement techniques than of physiological variations in parasites.…”

Section: Discussionmentioning

confidence: 91%

“…Furthermore, CQR parasites may have a more acidic DV pH [20]; this finding however has been debated [11,19,20,22,33]. Therefore, we measured the lysosomal pH in individual cells transfected with HB3-RFP and Dd2-RFP, compared with control cells transfected with RFP alone.…”

Section: Pfcrt Expression Caused Acidification Of the Ph In Lysosomesmentioning

confidence: 98%

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

Reeves

Liebelt

Lakshmanan

et al. 2006

Molecular and Biochemical Parasitology

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The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification of a Dd2 allele expressing the T76K "back mutation" was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca 2+ stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.

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“…To monitor hemoglobin uptake and parasite viability simultaneously we established a correlative flow cytometric assay employing parasites that had invaded resealed RBCs containing a fluorescent marker (21). The fluorescein-dextran was taken up into the parasite's digestive vacuole and used to monitor the level of endocytosis (green fluorescence intensity) following selective lysis of the RBC membrane by saponin (23).…”

Section: Resultsmentioning

confidence: 99%

Artemisinin activity againstPlasmodium falciparumrequires hemoglobin uptake and digestion

Klonis

Crespo-Ortiz²,

Bottová³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

316

314

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Combination regimens that include artemisinin derivatives are recommended as first line antimalarials in most countries where malaria is endemic. However, the mechanism of action of artemisinin is not fully understood and the usefulness of this drug class is threatened by reports of decreased parasite sensitivity. We treated Plasmodium falciparum for periods of a few hours to mimic clinical exposure to the short half-life artemisinins. We found that drug treatment retards parasite growth and inhibits uptake of hemoglobin, even at sublethal concentrations. We show that potent artemisinin activity is dependent on hemoglobin digestion by the parasite. Inhibition of hemoglobinase activity with cysteine protease inhibitors, knockout of the cysteine protease falcipain-2 by gene deletion, or direct deprivation of host cell lysate, significantly decreases artemisinin sensitivity. Hemoglobin digestion is also required for artemisinin-induced exacerbation of oxidative stress in the parasite cytoplasm. Arrest of hemoglobin digestion by early stage parasites provides a mechanism for surviving short-term artemisinin exposure. These insights will help in the design of new drugs and new treatment strategies to circumvent drug resistance.erythrocyte | endoperoxide

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The pH of the digestive vacuole ofPlasmodium falciparumis not associated with chloroquine resistance

Cited by 125 publications

References 79 publications

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

Artemisinin activity againstPlasmodium falciparumrequires hemoglobin uptake and digestion

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