Cecilia P. Sanchez

Hemoglobins S and C Interfere with Actin Remodeling in Plasmodium falciparum–Infected Erythrocytes

The malaria parasite mines actin from the membrane skeleton of its erythrocyte host to generate a cytoskeletal structure. The hemoglobins S and C protect carriers from severe Plasmodium falciparum malaria. Here, we found that these hemoglobinopathies affected the trafficking system that directs parasite-encoded proteins to the surface of infected erythrocytes. Cryoelectron tomography revealed that the parasite generated a host-derived actin cytoskeleton within the cytoplasm of wild-type red blood cells that connected the Maurer’s clefts with the host cell membrane and to which transport vesicles were attached. The actin cytoskeleton and the Maurer’s clefts were aberrant in erythrocytes containing hemoglobin S or C. Hemoglobin oxidation products, enriched in hemoglobin S and C erythrocytes, inhibited actin polymerization in vitro and may account for the protective role in malaria.

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

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 parasites 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 parasites food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed.

Transporters as mediators of drug resistance in Plasmodium falciparum.

Drug resistance represents a major obstacle in the radical control of malaria. Drug resistance can arise in many different ways, but recent developments highlight the importance of mutations in transporter molecules as being major contributors to drug resistance in the human malaria parasite Plasmodium falciparum. While approximately 2.5% of the P. falciparum genome encodes membrane transporters, this review concentrates on three transporters, namely the chloroquine resistance transporter PfCRT, the multi-drug resistance transporter 1 PfMDR1, and the multi-drug resistance-associated protein PfMRP, which have been strongly associated with resistance to the major antimalarial drugs. The studies that identified these entities as contributors to resistance, and the possible molecular mechanisms that can bring about this phenotype, are discussed. A deep understanding of the underpinning mechanisms, and of the structural specificities of the players themselves, is a necessary basis for the development of the new drugs that will be needed for the future armamentarium against malaria.

Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum.

Resistance to several anti‐malarial drugs has been associated with polymorphisms within the P‐glycoprotein homologue (Pgh‐1, PfMDR1) of the human malaria parasite Plasmodium falciparum. Pgh‐1, coded for by the gene pfmdr1, is predominately located at the membrane of the parasites digestive vacuole. How polymorphisms within this transporter mediate alter anti‐malarial drug responsiveness has remained obscure. Here we have functionally expressed pfmdr1 in Xenopus laevis oocytes. Our data demonstrate that Pgh‐1 transports vinblastine, an established substrate of mammalian MDR1, and the anti‐malarial drugs halofantrine, quinine and chloroquine. Importantly, polymorphisms within Pgh‐1 alter the substrate specificity for the anti‐malarial drugs. Wild‐type Pgh‐1 transports quinine and chloroquine, but not halofantrine, whereas polymorphic Pgh‐1 variants, associated with altered drug responsivenesses, transport halofantrine but not quinine and chloroquine. Our data further suggest that quinine acts as an inhibitor of Pgh‐1. Our data are discussed in terms of the model that Pgh‐1‐mediates, in a variant‐specific manner, import of certain drugs into the P. falciparum digestive vacuole, and that this contributes to accumulation of, and susceptibility to, the drug in question.

Identification of a Chloroquine Importer in Plasmodium falciparum DIFFERENCES IN IMPORT KINETICS ARE GENETICALLY LINKED WITH THE CHLOROQUINE-RESISTANT PHENOTYPE

We demonstrate that uptake of the antimalarial drug chloroquine is temperature-dependent, saturable, and inhibitable in Plasmodium falciparum. These features are indicative of carrier-mediated transport and suggest that a P. falciparum-encoded protein facilitates chloroquine import. Although both chloroquine-resistant and susceptible parasite isolates exhibit facilitated chloroquine uptake, the kinetics differ. Chloroquine-resistant parasite isolates consistently have an import mechanism with a lower transport activity and a reduced affinity for chloroquine. These differences in uptake kinetics are linked with chloroquine resistance in a genetic cross. These data suggest that changes in chloroquine import kinetics constitute a minimal and necessary event in the generation of the resistant phenotype. Competitive inhibition of chloroquine uptake by amiloride derivatives further suggests that chloroquine import is mediated by a plasmodial Na+/H+ exchanger.

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

Antimalarial dual drugs based on potent inhibitors of glutathione reductase from Plasmodium falciparum.

Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems providing a steady glutathione flux. As a future generation of dual drugs, 18 naphthoquinones and phenols (or their reduced forms) containing three different linkers between the 4-aminoquinoline core and the redox active component were synthesized. Their antimalarial effects have been characterized in parasite assays using chloroquine-sensitive and -resistant strains of Plasmodium, alone or in drug combination, and in the Plasmodium berghei rodent model. In particular, two tertiary amides 34 and 36 showed potent antimalarial activity in the low nanomolar range against CQ-resistant parasites. The ability to compete both for (Fe (III))protoporphyrin and for chloroquine transporter was determined. The data are consistent with the presence of a carrier for uptake of the short chloroquine analogue 2 but not for the potent antimalarial amide 34, suggesting a mode of action distinct from chloroquine mechanism.

Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasites food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans‐stimulation efflux protocol. While carriers may demonstrate trans‐stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine‐sensitive and ‐resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine‐resistant parasites. As trans‐stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine‐sensitive and ‐resistant parasites, however, with different transport modalities.

Illumination of the Malaria Parasite Plasmodium falciparum Alters Intracellular pH IMPLICATIONS FOR LIVE CELL IMAGING

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasitePlasmodium falciparum, including pH homeostasis, Ca2+ signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasites acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

Genetic Predisposition Favors the Acquisition of Stable Artemisinin Resistance in Malaria Parasites

ABSTRACT The emergence of artemisinin-resistant Plasmodium falciparum malaria jeopardizes efforts to control this infectious disease. To identify factors contributing to reduced artemisinin susceptibility, we have employed a classical genetic approach by analyzing artemisinin responses in the F1 progeny of a genetic cross. Our data show that reduced artemisinin susceptibility is a multifactorial trait, with pfmdr1 and two additional loci (on chromosomes 12 and 13) contributing to it. We further show that the different artemisinin susceptibilities of the progeny strains affect their responses to selection with increasing concentrations of artemisinin. Stable, high-level in vitro artemisinin resistance rapidly arose in those parasites that were the least artemisinin susceptible among the F1 progeny, whereas progeny that were highly artemisinin susceptible did not acquire stable artemisinin resistance. These data suggest that genetic predisposition favors the acquisition of high-level artemisinin resistance. In vitro-induced artemisinin resistance did not result in cross-resistance to artesunate or artemether, suggesting that resistance to one derivative does not necessarily render the entire drug class ineffective.