Kiaran Kirk

Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum

Throughout the latter half of this century, the development and spread of resistance to most front-line antimalarial compounds used in the prevention and treatment of the most severe form of human malaria has given cause for grave clinical concern. Polymorphisms in pfmdr1, the gene encoding the P-glycoprotein homologue 1 (Pgh1) protein of Plasmodium falciparum, have been linked to chloroquine resistance; Pgh1 has also been implicated in resistance to mefloquine and halofantrine. However, conclusive evidence of a direct causal association between pfmdr1 and resistance to these antimalarials has remained elusive, and a single genetic cross has suggested that Pgh1 is not involved in resistance to chloroquine and mefloquine. Here we provide direct proof that mutations in Pgh1 can confer resistance to mefloquine, quinine and halofantrine. The same mutations influence parasite resistance towards chloroquine in a strain-specific manner and the level of sensitivity to the structurally unrelated compound, artemisinin. This has important implications for the development and efficacy of future antimalarial agents.

Swelling-activated organic osmolyte channels.

Most cells have in their cytosols substantial (i.e., umillimolar) concentrations of low molecular weight organic solutes which, together, make a significant contribution to the total intracellular osmolality and which are known collectively as ‘organic osmolytes’. The solutes fulfilling this role fall, in most cases, into one of three different classes: amino acids (e.g., the a-amino acids alanine and proline, and the b-amino acids taurine and b-alanine), polyols (e.g., sorbitol and myo-inositol), and methylamines (e.g., betaine and glycerophosphoryl choline). Such compounds are either synthesized within the cell or taken up from the extracellular medium via accumulative (‘active’) transport systems. In contrast to inorganic ions which, at high concentrations, destabilize protein structure, these organic solutes exert a stabilizing influence on intracellular proteins and, for this reason, are termed ‘compatible solutes’ (Yancey, 1994). The identity and intracellular concentrations of the major organic osmolytes vary between cell-types, as well as with the conditions to which the cells are exposed. Intracellular levels of these compounds increase markedly in response to cell shrinkage. Conversely, a common phenomenon that has been demonstrated for a wide range of cells is that such compounds are released in response to an acute increase in the cell volume, as occurs, for example, following a sudden decrease in the extracellular osmolality. Their loss from the cell is accompanied by a net efflux of water and this process thereby serves as part of the cell’s volume-regulatory response. From studies of swelling-activated organic osmolyte transport in cells from a diverse range of organisms it has emerged that the transport pathways involved share a number of functional characteristics. There is increasing evidence that these pathways are, in many cases and perhaps in general, channels that have a significant permeability to a wide variety of both charged and uncharged solutes. The purpose of this review is to summarize what is currently known about these pathways in eukaryotic cells. The focus is on the properties of the pathways themselves. The mechanisms underlying their volume-sensitivity and the regulatory processes involved in their activation are not considered in any detail. Current ideas concerning the functional and molecular characteristics of the pathways are discussed. However, before turning to these issues it is relevant to consider the relative contribution that these pathways make to the process of ‘regulatory volume decrease’ (RVD) in different cell and tissue types.

pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase.

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H+ ions and thereby regulates its cytosolic pH (pH i ), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH i and to recover from an imposed intracellular acidification in the absence of extracellular Na+, thus ruling out the involvement of a Na+/H+ exchanger in both processes. Both phenomena were ATP-dependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH i of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na+/H+ exchange, had relatively little effect. The resting pH i of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blockerN,N′-dicyclohexylcarbodiimide, and by bafilomycin A1, a potent inhibitor of V-type H+-ATPases. Bafilomycin A1 blocked pH i recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H+-ATPase, which serves as the major route for the efflux of H+ ions.

Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance.

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra‐erythrocytic parasites digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil‐reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.

Coenzyme A biosynthesis: an antimicrobial drug target

Pantothenic acid, a precursor of coenzyme A (CoA), is essential for the growth of pathogenic microorganisms. Since the structure of pantothenic acid was determined, many analogues of this essential metabolite have been prepared. Several have been demonstrated to exert an antimicrobial effect against a range of microorganisms by inhibiting the utilization of pantothenic acid, validating pantothenic acid utilization as a potential novel antimicrobial drug target. This review commences with an overview of the mechanisms by which various microorganisms acquire the pantothenic acid they require for growth, and the universal CoA biosynthesis pathway by which pantothenic acid is converted into CoA. A detailed survey of studies that have investigated the inhibitory activity of analogues of pantothenic acid and other precursors of CoA follows. The potential of inhibitors of both pantothenic acid utilization and biosynthesis as novel antibacterial, antifungal and antimalarial agents is discussed, focusing on inhibitors and substrates of pantothenate kinase, the enzyme catalysing the rate-limiting step of CoA biosynthesis in many organisms. The best strategies are considered for identifying inhibitors of pantothenic acid utilization and biosynthesis that are potent and selective inhibitors of microbial growth and that may be suitable for use as chemotherapeutic agents in humans.

The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

BackgroundThe uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these.ResultsA computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared.ConclusionThe malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen.

A surface transporter family conveys the trypanosome differentiation signal.

Microbial pathogens use environmental cues to trigger the developmental events needed to infect mammalian hosts or transmit to disease vectors. The parasites causing African sleeping sickness respond to citrate or cis-aconitate (CCA) to initiate life-cycle development when transmitted to their tsetse fly vector. This requires hypersensitization of the parasites to CCA by exposure to low temperature, conditions encountered after tsetse fly feeding at dusk or dawn. Here we identify a carboxylate-transporter family, PAD (proteins associated with differentiation), required for perception of this differentiation signal. Consistent with predictions for the response of trypanosomes to CCA, PAD proteins are expressed on the surface of the transmission-competent ‘stumpy-form’ parasites in the bloodstream, and at least one member is thermoregulated, showing elevated expression and surface access at low temperature. Moreover, RNA-interference-mediated ablation of PAD expression diminishes CCA-induced differentiation and eliminates CCA hypersensitivity under cold-shock conditions. As well as being molecular transducers of the differentiation signal in these parasites, PAD proteins provide the first example of a surface marker able to discriminate the transmission stage of trypanosomes in their mammalian host.

Calothrixins A and B, novel pentacyclic metabolites from Calothrix cyanobacteria with potent activity against malaria parasites and human cancer cells

Abstract Cell extracts from photoautrophic cultures of two cyanobacterial Calothrix isolates inhibited the growth in vitro of a chloroquine-resistant strain of the malaria parasite, Plasmodium falciparum, and of human HeLa cancer cells, in a dose-dependent manner. Bioassay-directed fractionation of the extracts led to the isolation and structural characterization of calothrixins A (1) and B (2), pentacyclic metabolites with an indolo[3,2-j]phenanthridine ring system unique amongst natural products, which exert their growth-inhibitory effects at nanomolar concentrations.

Na+ Regulation in the Malaria Parasite Plasmodium falciparum Involves the Cation ATPase PfATP4 and Is a Target of the Spiroindolone Antimalarials

Summary The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na+ influx, causing [Na+] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na+] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na+ against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na+-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na+ homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na+ regulation. Taken together, our results are consistent with PfATP4 being a Na+ efflux ATPase and a target of the spiroindolones.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication. Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na+ homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.