Increased permeability of the malaria-infected erythrocyte to organic cations

AbstractThe human malaria parasite, Plasmodium falciparum, induces in the plasma membrane of its host red blood cell new permeation pathways (NPP) that allow the influx of a variety of low molecular weight solutes. In this study we have demonstrated that the NPP confer upon the parasitised erythrocyte a substantial permeability to a range of monovalent organic (quaternary ammonium) cations, the largest having an estimated minimum cross-sectional diameter of 11–12 Å. The rate of permeation of these cations showed a marked dependence on the nature of the anion present, increasing with the lyotropicity of the anion. There was no clear relationship between the permeation rate and either the size or the hydrophobicity of these solutes. However, the data were consistent with the rate of permeation being influenced by a combination of these two factors, with the pathways showing a marked preference for the relatively small and hydrophobic phenyltrimethylammonium ion over larger or less hydrophobic solutes. Large quaternary ammonium cations inhibited flux via the NPP, as did long-chain n-alkanols. For both classes of compound the inhibitory potency increased with the size and hydrophobicity of the solute. This study extends the range of solutes known to permeate the NPP of malaria-infected erythrocytes as well as providing some insight into the factors governing the rate of permeation

Selective Inhibition of Plasmodium falciparum ATPase 6 by Artemisinins and Identification of New Classes of Inhibitors after Expression in Yeast

Treatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work. Here, we report the successful development of a heterologous expression-based compound-screening tool. The validated drug target Plasmodium falciparum ATPase 6 (PfATP6) and a mammalian orthologue (sarco/endoplasmic reticulum calcium ATPase 1a [SERCA1a]) were functionally expressed in Saccharomyces cerevisiae, providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labeling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model. As previously hypothesized, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low-technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multitargeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility

An efficient and novel technology for the extraction of parasite genomic DNA from whole blood or culture

The aim of this study was to assess pathogen DNA extraction with a new spin column-based method (DNA-XT). DNA from either whole-blood samples spiked with Plasmodium falciparum or Leishmania donovani amastigote culture was extracted with DNA-XT and compared with that produced by a commercial extraction kit (DNeasy®). Eluates from large and small sample volumes were assessed by PCR and spectroscopy. Using a small volume (5 μl) of blood, the DNA-XT and DNeasy methods produced eluates with similar DNA concentrations (0.63 vs 1.06 ng/μl, respectively). The DNA-XT method produced DNA with lower PCR inhibition than DNeasy. The new technique was also twice as fast and required fewer plastics and manipulations but had reduced total recovered DNA compared with DNeasy

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcom

Adaptation of in vitro cytoadherence assay to Plasmodium knowlesi field isolates

P. knowlesi was the first Plasmodium species in which antigenic variation was observed. Variation was due to schizont infected cell agglutination (SICAvar) antigens expressed by the parasite and transported to the exposed surface of the host erythrocyte [1]. PfEMP1 is P. falciparum’s orthologue of P. knowlesi’s SICA proteins [2]. In P. falciparum PfEMP1 is associated with infected erythrocytes binding to receptors such as ICAM-1 expressed on the endothelial cells of the host microvasculature. Here, we use a static protein assay [3] to determine if naturally occurring human P. knowlesi infections can cause erythrocytes to bind to ICAM-1, VCAM-1 and CD36

An efficient and novel technology for the extraction of parasite genomic DNA from whole blood or culture

The aim of this study was to assess pathogen DNA extraction with a new spin column-based method (DNA-XT). DNA from either whole-blood samples spiked with Plasmodium falciparum or Leishmania donovani amastigote culture was extracted with DNA-XT and compared with that produced by a commercial extraction kit (DNeasy®). Eluates from large and small sample volumes were assessed by PCR and spectroscopy. Using a small volume (5 μl) of blood, the DNA-XT and DNeasy methods produced eluates with similar DNA concentrations (0.63 vs 1.06 ng/μl, respectively). The DNA-XT method produced DNA with lower PCR inhibition than DNeasy. The new technique was also twice as fast and required fewer plastics and manipulations but had reduced total recovered DNA compared with DNeasy

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum

A recent study on malaria-infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host- or parasite-derived and their molecular nature have not been resolved. Here we report a comparison of a malaria-induced anion channel with an endogenous anion channel in Plasmodium falciparum-infected human RBCs. Ion channel activity was measured using the whole-cell, cell-attached and excised inside-out configurations of the patch-clamp method. Parasitised RBCs were cultured in vitro, using co-cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (~15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I(-) > Br(-) > Cl(-). In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (~80 pS, outward conductance) was spontaneously active. The host membrane of malaria-infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria-infected RBCs were shown to have a low open-state probability (P(o)) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole-cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up-regulated by the malaria parasite P. falciparum

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs

Susceptibility of human Plasmodium knowlesi infections to anti-malarials

Background: Evidence suggests that Plasmodium knowlesi malaria in Sarawak, Malaysian Borneo remains zoonotic, meaning anti-malarial drug resistance is unlikely to have developed in the absence of drug selection pressure. Therefore, adequate response to available anti-malarial treatments is assumed. Methods: Here the ex vivo sensitivity of human P. knowlesi isolates in Malaysian Borneo were studied, using a WHO schizont maturation assay modified to accommodate the quotidian life cycle of this parasite. The in vitro sensitivities of P. knowlesi H strain adapted from a primate infection to in vitro culture (by measuring the production of Plasmodium lactate dehydrogenase) were also examined together with some assays using Plasmodium falciparum and Plasmodium vivax. Results: Plasmodium knowlesi is uniformly highly sensitive to artemisinins, variably and moderately sensitive to chloroquine, and less sensitive to mefloquine. Conclusions: Taken together with reports of clinical failures when P. knowlesi is treated with mefloquine, the data suggest that caution is required if using mefloquine in prevention or treatment of P. knowlesi infections, until further studies are undertaken

Plasmodial sugar transporters as anti-malarial drug targets and comparisons with other protozoa

Glucose is the primary source of energy and a key substrate for most cells. Inhibition of cellular glucose uptake (the first step in its utilization) has, therefore, received attention as a potential therapeutic strategy to treat various unrelated diseases including malaria and cancers. For malaria, blood forms of parasites rely almost entirely on glycolysis for energy production and, without energy stores, they are dependent on the constant uptake of glucose. Plasmodium falciparum is the most dangerous human malarial parasite and its hexose transporter has been identified as being the major glucose transporter. In this review, recent progress regarding the validation and development of the P. falciparum hexose transporter as a drug target is described, highlighting the importance of robust target validation through both chemical and genetic methods. Therapeutic targeting potential of hexose transporters of other protozoan pathogens is also reviewed and discussed