Mutations Conferring Drug Resistance in Malaria Parasite Drug Transporters Pgh1 and PfCRT Do Not Affect Steady-State Vacuolar Ca
            <sup>2+</sup>

Biagini, Giancarlo A.; Fidock, David A.; Bray, Patrick G.; Ward, Stephen A.

doi:10.1128/aac.49.11.4807-4808.2005

Cited by 11 publications

(7 citation statements)

References 20 publications

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“…No significant differences in cytoplasmic or food vacuolar free [Ca 2+ ] i were found between the examined parasites Dd2, K1, HB3 and NF54 ( P >0.05) (Table I). The free [Ca 2+ ] i values measured were consistent with previous determinations (Biagini et al , 2003, 2005; Rohrbach et al , 2005).…”

Section: Resultssupporting

confidence: 91%

“…Our quantitative Ca 2+ determinations relied on Fura‐Red AM, a ratiometric Ca 2+ indicator that provides reliable recordings of steady‐state free [Ca 2+ ] i in different subcellular compartments of P. falciparum ‐infected erythrocytes (Rohrbach et al , 2005). The steady‐state food vacuolar and cytoplasmic free [Ca 2+ ] i values reported herein are consistent with previous determinations (Biagini et al , 2003, 2005; Rohrbach et al , 2005).…”

Section: Discussionsupporting

confidence: 92%

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Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

Rohrbach

Sánchez

Hayton

et al. 2006

EMBO J

127

149

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The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential staining of the food vacuole versus a more diffuse staining of the entire parasite. Genetic, positional cloning and pharmacological data causatively link the food vacuolar Fluo-4 phenotype to those Pgh-1 variants that are associated with altered drug responses. On the basis of our data, we propose that Pgh-1 imports solutes, including certain antimalarial drugs, into the parasite's food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed.

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Section: Resultssupporting

confidence: 91%

Section: Discussionsupporting

confidence: 92%

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

Rohrbach

Sánchez

Hayton

et al. 2006

EMBO J

127

149

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“…The changes in intralysosomal pH associated with PfCRT expression were not due to a generalized change in lysosomal ion transport, as we detected no significant difference between the peak lysosomal Ca 2+ release induced by GPN in mock-transfected cells compared with either HB3-RFP-or Dd2-RFP-transfected cells, an observation consistent with that obtained for the parasite DV [50].…”

Section: Discussionsupporting

confidence: 90%

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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“…An equal volume of EBSS containing only 48 nM [ 3 H]-GSH (2 µCi ml −1 , American Radiolabelled Chemicals, USA) or 48 nM [ 3 H]-GSH and 6 mM cold GSH was added and carefully mixed, giving final concentrations of 24 nM [ 3 H]-GSH and 3 mM cold GSH. The cells were incubated in a water bath at 37°C and at various time points 200 µl samples corresponding to 2 × 10 7 RBC were removed and cells were immediately separated from the uptake solution by centrifugation through 300 µl dibutylphthalate as described in Biagini et al . (2005) and Quashie et al .…”

Section: Methodsmentioning

confidence: 99%

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Patzewitz

Wong

Müller

2011

Molecular Microbiology

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Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.

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Mutations Conferring Drug Resistance in Malaria Parasite Drug Transporters Pgh1 and PfCRT Do Not Affect Steady-State Vacuolar Ca ²⁺

Cited by 11 publications

References 20 publications

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

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

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

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Mutations Conferring Drug Resistance in Malaria Parasite Drug Transporters Pgh1 and PfCRT Do Not Affect Steady-State Vacuolar Ca 2+

Cited by 11 publications

References 20 publications

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

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

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Contact Info

Product

Resources

About

Mutations Conferring Drug Resistance in Malaria Parasite Drug Transporters Pgh1 and PfCRT Do Not Affect Steady-State Vacuolar Ca ²⁺