Evolutionary paradigm of chloroquine-resistant malaria in India

Drug pressure in the field is believed to be responsible for the emergence of drug-resistant Plasmodium falciparum, the parasite that causes malaria. Variants of the P. falciparum chloroquine resistance transporter (pfcrt) gene have been shown to be responsible for conferring resistance to the commonly used drug chloroquine. In particular, an amino acid mutation, K76T, was shown to have a strong positive correlation with the chloroquine-resistant varieties of malaria parasites. Global studies have reported highly reduced genetic diversity surrounding K76T in the pfcrt gene, which indicates that the mutation has been a target of positive Darwinian natural selection. However, two recent studies of P. falciparum in India found high genetic diversity in the pfcrt gene, which, at first sight, do not support the role of natural selection in the evolution of chloroquine resistance in India.     

Environmental, pharmacological and genetic influences on the spread of drug-resistant malaria

Plasmodium falciparum malaria is subject to artificial selection from antimalarial drugs that select for drug-resistant parasites. We describe and apply a flexible new approach to investigate how epistasis, inbreeding, selection heterogeneity and multiple simultaneous drug deployments interact to influence the spread of drug-resistant malaria. This framework recognizes that different human 'environments' within which treatment may occur (such as semi- and non-immune humans taking full or partial drug courses) influence the genetic interactions between parasite loci involved in resistance. Our model provides an explanation for how the rate of spread varies according to different malaria transmission intensities, why resistance might stabilize at intermediate frequencies and also identifies several factors that influence the decline of resistance after a drug is removed. Results suggest that studies based on clinical outcomes might overestimate the spread of resistant parasites, especially in high-transmission areas. We show that when transmission decreases, prevalence might decrease without a corresponding change in frequency of resistance and that this relationship is heavily influenced by the extent of linkage disequilibrium between loci. This has important consequences on the interpretation of data from areas where control is being successful and suggests that reducing transmission might have less impact on the spread of resistance than previously expected.     

Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites

Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or “Accelerated Resistance to Multiple Drugs” (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na⁺/H⁺-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring detrimental fitness costs.     

The influence of drug treatment on the maintenance of schistosome genetic diversity

Drug treatment of patients with schistosomiasis may select for drug-resistant parasites. In this article, we formulate a deterministic model with multiple strains of schistosomes (helminth parasites with a two-host life cycles) in order to explore the role of drug treatment in the maintenance of a polymorphism of parasite strains that differ in their resistance levels. The basic reproductive numbers for all strains are computed, and are shown to determine the stabilities of equilibria of the model and consequently the distribution of parasite phenotypes with different levels of drug tolerance. Analysis of our model shows that the likelihood that resistant strains will increase in frequency depends on the interplay between their relative fitness, the cost of resistance, and the degree of selection pressure exerted by the drug treatments.     

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Mechanisms of drug resistance in Leishmania

The emergence of drug resistance in protozoan parasites is a major obstacle to their control. Since vaccines are not yet in sight for several of these parasites, there is on urgent need to develop new and better drugs. These antimicrobial agents will possibly be more expensive, and will therefore impose on additional burden in health-care costs and in the planning of public health policies of the developing countries. A better understanding of drug resistance, to try to circumvent or overcome it, and the search for new specific cellular targets of parasites are warranted. The development, in vitro, of drug-resistant parasite cell lines has been instrumental in our understanding of the mechanisms of drug resistance in parasitic protozoans. Marc Ouellette and Barbara Popodopoulou here present on overview of the recent progress on the elucidation of mechanisms of drug resistance in the protozoan parasite Leishmania, selected under laboratory conditions.     

Superinfection and the evolution of resistance to antimalarial drugs

A major issue in the control of malaria is the evolution of drug resistance. Ecological theory has demonstrated that pathogen superinfection and the resulting within-host competition influences the evolution of specific traits. Individuals infected with Plasmodium falciparum are consistently infected by multiple parasites; however, while this probably alters the dynamics of resistance evolution, there are few robust mathematical models examining this issue. We developed a general theory for modelling the evolution of resistance with host superinfection and examine: (i) the effect of transmission intensity on the rate of resistance evolution; (ii) the importance of different biological costs of resistance; and (iii) the best measure of the frequency of resistance. We find that within-host competition retards the ability and slows the rate at which drug-resistant parasites invade, particularly as the transmission rate increases. We also find that biological costs of resistance that reduce transmission are less important than reductions in the duration of drug-resistant infections. Lastly, we find that random sampling of the population for resistant parasites is likely to significantly underestimate the frequency of resistance. Considering superinfection in mathematical models of antimalarial drug resistance may thus be important for generating accurate predictions of interventions to contain resistance.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.     

An Analytical Method for Assessing Stage-Specific Drug Activity in Plasmodium vivax Malaria: Implications for Ex Vivo Drug Susceptibility Testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC(50)) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC(50) for CQ. Our findings indicate that EC(50) values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.     

Plasmodium falciparum: cloning by single-erythrocyte micromanipulation and heterogeneity in vitro

Natural isolates of Plasmodium falciparum represent a genetically heterogeneous population of parasites. To obtain stable strains of the parasites for long term experiments, a rapid and definitive method of cloning was developed using micropipets and a micromanipulator. Homogeneous parasite clones prepared by this technique were characterized and compared with the parent isolates during 4 years of continuous culture. The process of phenotypic dominance and selection of drug resistance which occur in nature was also simulated in vitro by evaluating population dynamics of two cocultured isolates of P. falciparum.     

Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite.

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.     

Quantification of multiple infections of Plasmodium falciparum in vitro

ABSTRACT: BACKGROUND: Human malaria infections caused by the parasite Plasmodium falciparum often contain more than one genetically distinct parasite. Despite this fact, nearly all studies of multiple strain P. falciparum infections have been limited to determining relative densities of each parasite within an infection. In light of this, new methods are needed that can quantify the absolute number of parasites within a single infection. METHODS: A quantitative PCR (qPCR) method was developed to track the dynamic interaction of P. falciparum infections containing genetically distinct parasite clones in cultured red blood cells. Allele-specific primers were used to generate a standard curve and to quantify the absolute concentration of parasite DNA within multi-clonal infections. Effects on dynamic growth relationships between parasites under drug pressure were examined by treating mixed cultures of drug sensitive and drug resistant parasites with the anti-malarial drug chloroquine at different dosing schedules. RESULTS: An absolute quantification method was developed to monitor the dynamics of P. falciparum cultures in vitro. This method allowed for the observation of competitive suppression, the reduction of parasites numbers due to the presence of another parasite, and competitive release, the improved performance of a parasite after the removal of a competitor. These studies demonstrated that the presence of two parasites led to the reduction in density of at least one parasite. containing both a drug resistant and drug sensitive parasites resulted in an increased proportion of the drug resistant parasite. Moreover, following drug treatment, the resistant parasite experienced competitive release by exhibiting a fitness benefit greater than simply surviving drug treatment, due to the removal of competitive suppression by the sensitive parasite. CONCLUSIONS: The newly developed assay allowed for the examination of the dynamics of two distinct clones in vitro; both competitive suppression and release were observed. A deeper understanding of the dynamic growth responses of multiple strain P. falciparum infections, with and without drug pressure, can improve the understanding of the role of parasite interactions in the spread of drug resistant parasites, perhaps suggesting different treatment strategies.     

Cysteine protease inhibitors as chemotherapy for parasitic infections.

Analysis of the evolution, localization and biologic function of papain family cysteine proteases in metazoan and protozoan parasites has provided important and often surprising insights into the biochemistry and cellular function of this diverse enzyme family. Furthermore, the relative lack of redundancy of cysteine proteases in parasites compared to their mammalian hosts makes them attractive targets for the development of new antiparasitic chemotherapy. The treatment of experimental models of parasitic diseases with cysteine protease inhibitors has provided an important 'proof of concept' for the use of cysteine protease inhibitors in vivo. Evidence has now accumulated that cysteine protease inhibitors can selectively arrest replication of a microbial pathogen without untoward toxicity to the host. Furthermore, this can be achieved with reasonable dosing schedules and oral administration of the drug. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. Work on Trypanosoma brucei, the agent of African trypanosomiasis, is preliminary but also promising. Target validation studies have shown that biotinylated or radiolabeled irreversible inhibitors specifically bind to the cysteine protease targets thought to represent the major activity within the parasite. In the case of T. cruzi, the effect of inhibitors appears to be predominantly in blocking protease processing. Transfection studies using variant constructs have supported this model. Finally, the generation of null mutants for the multiple protease genes in Leishmania mexicana has provided the first genetic support for the key role of this enzyme family in parasite virulence. Safety studies in rodents and analysis of uptake of inhibitors by parasites and host cells suggest that the selectivity of inhibitors for the parasite targets may reside in the lack of redundancy of parasite proteases, the higher concentration of host proteases in intracellular compartments, and differential uptake of inhibitors by parasites. Attempts to elicit resistance to cysteine protease inhibitors in parasite cultures suggest that mechanisms of induced resistance are independent of resistance to the traditional antiparasitic agents. This suggests that cysteine protease inhibitors may provide an alternative to traditional therapy in drug-resistant organisms.     

Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.