Genetic and genomic approaches for the discovery of parasite genes involved in antimalarial drug resistance

The biggest threat to the war on malaria is the continued evolution of drug resistance by the parasite. Resistance to almost all currently available antimalarials now exists in Plasmodium falciparum which causes the most suffering among all human malaria parasites. Monitoring of antimalarial efficacy and the development and subsequent spread of resistance has become an important part in the treatment and control of malaria. With recent reports of reduced efficacy of artemisinin, the current recommended treatment for uncomplicated malaria, there is urgent need for better methods to recognize and monitor drug resistance for effective treatment. Molecular markers have become a welcome addition to complement the more laborious and costly in vitro and in vivo methods that have traditionally been used to monitor drug resistance. However, there are currently no molecular markers for resistance to some antimalarials. This review highlights the role of the various genetic and genomic approaches that have been used in identifying the molecular markers that underlie drug resistance in P. falciparum. These approaches include; candidate genes, genetic linkage and genome-wide association studies. We discuss the requirements and limitations of each approach and use various examples to illustrate their contributions in identifying genomic regions of the parasite associated with antimalarial drug responses

Uptake of purines in <i>Plasmodium falciparum</i>-infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter

&lt;b&gt;Background&lt;/b&gt;: Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. &lt;b&gt;Methods&lt;/b&gt;: The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied. &lt;b&gt;Results&lt;/b&gt;: Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1. &lt;b&gt;Conclusion&lt;/b&gt;: Collectively, the data obtained in this study clearly show that the endogenous host erythrocyte transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport

The impact of uniform and mixed species blood meals on the fitness of the mosquito vector Anopheles gambiae s.s: does a specialist pay for diversifying its host species diet?

We investigated the fitness consequences of specialization in an organism whose host choice has an immense impact on human health: the African malaria vector Anopheles gambiae s.s. We tested whether this mosquito’s specialism on humans can be attributed to the relative fitness benefits of specialist vs. generalist feeding strategies by contrasting their fecundity and survival on human-only and mixed host diets consisting of blood meals from humans and animals. When given only one blood meal, An. gambiae s.s. survived significantly longer on human and bovine blood, than on canine or avian blood. However, when blood fed repeatedly, there was no evidence that the fitness of An. gambiae s.s. fed a human-only diet was greater than those fed generalist diets. This suggests that the adoption of generalist host feeding strategies in An. gambiae s.s. is not constrained by intraspecific variation in the resource quality of blood from other available host species

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocol

Fit for fertilization: mating in malaria parasites

The human malaria parasite Plasmodium falciparum has an obligate sexual phase in its life cycle. Male and female gametes must mate in the mosquito midgut for transmission to occur. When mosquitoes ingest a mixture of two parasite clones, the inheritance of nuclear genes suggests that mating between gametes is random. Both cross-fertilization (between unlike male and female gametes) and selfing occur. However, it has been suggested that the inheritance of mitochondrial markers indicates non-random mating. An alternative hypothesis, which is presented here by Lisa Ranford-Cartwright, is that mating is random, but differences in the relative fitnesses of the gametocytes can explain the inheritance patterns observed

Genetic recombination in field populations of Plasmodium falciparum

Malaria parasites undergo a mainly haploid life-cycle. The only diploid stage is the zygote, formed by fusion of gametes in the mosquito stomach. The first division of the zygote is a meiotic one, producing, after further mitotic divisions, haploid sporozoites. Genetic recombination occurs at meiosis, following cross-fertilization of gametes of parasites with different genotypes. This has been shown in laboratory studies by feeding mosquitoes on a mixture of Plasmodium falciparum clones and analyzing the resulting progeny for parasites with non-parental combinations of the clone markers. Such recombinants are produced at a higher than expected frequency. There is considerable genotype diversity in field populations of P. falciparum. Evidence that recombination in mosquitoes is the principal cause of this diversity is two-fold. First, parasites isolated from patients in small isolated communities at the same time are genetically very diverse. No two isolates examined for polymorphic markers at some 20 loci have been found to possess identical combinations of the allelic variants of these genes. Second, examination of oocysts in wild-caught mosquitoes by the PCR technique has shown that a high proportion are heterozygotes. There is thus frequent crossing in natural populations of this parasite. In addition to recombination at meiosis, it is also clear that genetic changes can occur during asexual multiplication of P. falciparum blood forms, as shown by deletions of regions of certain chromosomes during in vitro culture. The extent to which this occurs in nature is not known

The role of asymptomatic P. falciparum parasitaemia in the evolution of antimalarial drug resistance in areas of seasonal transmission

In areas with seasonal transmission, proper management of acute malaria cases that arise in the transmission season can markedly reduce the disease burden. However, asymptomatic carriage of Plasmodium falciparum sustains a long-lasting reservoir in the transmission-free dry season that seeds cyclical malaria outbreaks. Clinical trials targeting asymptomatic parasitaemia in the dry season failed to interrupt the malaria epidemics that follow annual rains. These asymptomatic infections tend to carry multiple-clones, capable of producing gametocytes and infecting Anopheles mosquitoes. Different clones within an infection fluctuate consistently, indicative of interaction between clones during the long course of asymptomatic carriage. However, the therapy-free environment that prevails in the dry season dis-advantages the drug resistant lineages and favors the wild-type parasites. This review highlights some biological and epidemiological characteristics of asymptomatic parasitaemia and calls for consideration of polices to diminish parasite exposure to drugs “therapy-free” and allow natural selection to curb drug resistance in the above setting

Genetic recombination in field populations of Plasmodium falciparum

Malaria parasites undergo a mainly haploid life-cycle. The only diploid stage is the zygote, formed by fusion of gametes in the mosquito stomach. The first division of the zygote is a meiotic one, producing, after further mitotic divisions, haploid sporozoites. Genetic recombination occurs at meiosis, following cross-fertilization of gametes of parasites with different genotypes. This has been shown in laboratory studies by feeding mosquitoes on a mixture of Plasmodium falciparum clones and analyzing the resulting progeny for parasites with non-parental combinations of the clone markers. Such recombinants are produced at a higher than expected frequency. There is considerable genotype diversity in field populations of P. falciparum. Evidence that recombination in mosquitoes is the principal cause of this diversity is two-fold. First, parasites isolated from patients in small isolated communities at the same time are genetically very diverse. No two isolates examined for polymorphic markers at some 20 loci have been found to possess identical combinations of the allelic variants of these genes. Second, examination of oocysts in wild-caught mosquitoes by the PCR technique has shown that a high proportion are heterozygotes. There is thus frequent crossing in natural populations of this parasite. In addition to recombination at meiosis, it is also clear that genetic changes can occur during asexual multiplication of P. falciparum blood forms, as shown by deletions of regions of certain chromosomes during in vitro culture. The extent to which this occurs in nature is not known

Frequency of cross-fertilization in the human malaria parasite Plasmodium falciparum

Two clones of the human malaria parasite Plasmodium falciparum, denoted 3D7 and HB3, were grown in vitro under conditions permitting the development of gametocytes. The two clones differ in their allelic forms of two antigen genes MSP1 and MSP2. The alleles can be distinguished as size differences of polymerase chain reaction (PCR) amplified fragments of repetitive regions of each gene. Mosquitoes (Anopheles stephensi) were fed on a mixture of these gametocytes. A total of 128 oocysts was isolated from the midguts of infected mosquitoes from 9 crossing experiments between the clones. DNA extracted from these oocysts was amplified by PCR. Oocysts which contained both alleles of each gene (MSP1 and MSP2) had developed from heterozygotes produced by cross-fertilization events between 3D7 and HB3 gametes. The remaining oocysts contained single alleles of each gene, in parent clone combinations, and these had developed from homozygotes formed by self-fertilizations. The results suggest that gametes in the original mixture fed to mosquitoes had undergone random mating