Tetracysteine-based fluorescent tags to study protein localization and trafficking in Plasmodium falciparum-infected erythrocytes.

UNLABELLED: Plasmodium falciparum (Pf) malaria parasites remodel host erythrocytes by placing membranous structures in the host cell cytoplasm and inserting proteins into the surrounding erythrocyte membranes. Dynamic imaging techniques with high spatial and temporal resolutions are required to study the trafficking pathways of proteins and the time courses of their delivery to the host erythrocyte membrane. METHODOLOGY AND FINDINGS: Using a tetracysteine (TC) motif tag and TC-binding biarsenical fluorophores (BAFs) including fluorescein arsenical hairpin (FlAsH) and resorufin arsenical hairpin (ReAsH), we detected knob-associated histidine-rich protein (KAHRP) constructs in Pf-parasitized erythrocytes and compared their fluorescence signals to those of GFP (green fluorescent protein)-tagged KAHRP. Rigorous treatment with BAL (2, 3 dimercaptopropanol; British anti-Lewisite) was required to reduce high background due to nonspecific BAF interactions with endogenous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. The fluorescence from FlAsH and ReAsH-labeled protein bleached at faster rates than the fluorescence from GFP-labeled protein. CONCLUSION: While TC/BAF labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods. Our observations are in agreement with the currently-accepted model of KAHRP movement through the cytoplasm, including transient association of KAHRP with Maurer's clefts before its incorporation into knobs in the host erythrocyte membrane.

Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine.

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum originated from at least six foci in South America, Asia, and Oceania. Malaria parasites from these locations exhibit contrasting resistance phenotypes that are distinguished by point mutations and microsatellite polymorphisms in and near the CQR transporter gene, pfcrt, and the multidrug resistance transporter gene, pfmdr1. Amodiaquine (AQ), a 4-aminoquinoline related to CQ, is recommended and often used successfully against CQ-resistant P. falciparum in Africa, but it is largely ineffective across large regions of South America. The relationship of different pfcrt and pfmdr1 combinations to these drug-resistant phenotypes has been unclear. In two P. falciparum genetic crosses, particular pfcrt and pfmdr1 alleles from South America interact to yield greater levels of resistance to monodesethylamodiaquine (MDAQ; the active metabolite of AQ) than to CQ, whereas a pfcrt allele from Southeast Asia and Africa is linked to greater CQ than MDAQ resistance with all partner pfmdr1 alleles. These results, together with (i) available haplotype data from other parasites; (ii) evidence for an emerging focus of AQ resistance in Tanzania; and (iii) the persistence of 4-aminoquinoline-resistant parasites in South America, where CQ and AQ use is largely discontinued, suggest that different histories of drug use on the two continents have driven the selection of distinct suites of pfcrt and pfmdr1 mutations. Increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ-resistant, CQ-resistant parasites with pfcrt and pfmdr1 mutations that are as advantaged and persistent as in South America.

Expression and function of pvcrt-o, a Plasmodium vivax ortholog of pfcrt, in Plasmodium falciparum and Dictyostelium discoideum.

Chloroquine resistance in Plasmodium vivax threatens the use of this drug as first-line treatment for millions of people infected each year worldwide. Unlike Plasmodium falciparum, in which chloroquine resistance is associated with mutations in the pfcrt gene encoding a digestive vacuole transmembrane protein, no point mutations have been associated with chloroquine resistance in the P. vivax ortholog gene, pvcrt-o (also called pvcg10). However, the question remains whether pvcrt-o can affect chloroquine response independent of mutations. Since P. vivax cannot be cultured in vitro, we used two heterologous expression systems to address this question. Results from the first system, in which chloroquine sensitive P. falciparum parasites were transformed with pvcrt-o, showed a 2.2-fold increase in chloroquine tolerance with pvcrt-o expression under a strong promoter; this effect was reversed by verapamil. In the second system, wild type pvcrt-o or a mutated form of the gene was expressed in Dictyostelium discoideum. Forms of PvCRT-o engineered to express either lysine or threonine at position 76 produced a verapamil-reversible reduction of chloroquine accumulation in this system to approximately 60% of that in control cells. Our data support an effect of PvCRT-o on chloroquine transport and/or accumulation by P. vivax, independent of the K76T amino acid substitution.

Plasmodium vivax: allele variants of the mdr1 gene do not associate with chloroquine resistance among isolates from Brazil, Papua, and monkey-adapted strains.

We describe here the sequence of the Plasmodium vivax mdr1 gene from 10 different isolates differing in chloroquine sensitivity. The deduced amino acid sequence of PvMDR1 shares more than 70% similarity with other malarial MDR proteins and it displays consensus motifs of an ABC family transporter including two transmembrane domains and two ATP binding cassettes. Similarity and dendrogram analyses revealed that sequences could be grouped according to their geographical origin. Within each geographical group however, no correlation was found between chloroquine resistance and specific mutations.

Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity.

BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism. The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s. The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106-->Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process.