Margery Sullivan

Disruption of a Plasmodium falciparum Multidrug Resistance-associated Protein (PfMRP) Alters Its Fitness and Transport of Antimalarial Drugs and Glutathione

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Antimalarial drugs: current status and new developments

Malaria continues to be a major threat in the developing world, with > 1 million clinical episodes and 3000 deaths every day. In the last century, malaria claimed between 150 and 300 million lives, accounting for 2 – 5% of all deaths. Currently ~ 40% of the world population resides in areas of active malaria transmission. The disease symptoms are most severe in young children and pregnant women. A total of 90% of the disease-associated mortality occurs in Subsaharan Africa, despite the fact that malaria is indigenous to most tropical regions. A licensed vaccine for malaria has not become a reality and antimalarial drugs are the only available method of treatment. Although chloroquine, the first synthetically developed antimalarial, proved to be an almost magical cure for > 30 years, the emergence and spread of chloroquine-resistant parasites has made it virtually ineffective in most parts of the world. Currently, artemisinin, a plant-derived antimalarial, is the only available drug that is globally effective against the parasite. Although several new drugs have been introduced in the past 30 years, widespread or isolated cases of resistance indicate that their window of effectiveness will be limited. Thus, there is an urgent need to develop new therapeutics and regimens for malaria control. This article presents an overview of the currently available antimalarial chemotherapy options and the efforts being undertaken to develop new drugs based on both the recent technological advances and modifications to the old remedies, and on combination therapies.

A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species

Gene trees of Plasmodium species have been reported for the nuclear encoded genes (e.g. the Small Subunit rRNA) and a mitochondrial encoded gene, cytochrome b. Here, we have analyzed a plastid gene coding for caseinolytic protease ClpC, whose structure, function and evolutionary history have been studied in various organisms. This protein possesses a 220-250 amino acid long AAA domain (ATPases associated with a variety of cellular activities) that belongs to the Walker super family of ATPases and GTPases. We have sequenced the AAA motif of this gene, encoding the protein from nine different species of Plasmodium infecting rodents, birds, monkeys, and humans. The codon usage and GC content of each gene were nearly identical in contrast to the widely varying nucleotide composition of genomic DNAs. Phylogenetic trees derived from both DNA and inferred protein sequences have consistent topologies. We have used the ClpC sequence to analyze the phylogenetic relationship among Plasmodium species and compared it with those derived from mitochondrial and genomic sequences. The results corroborate well with the trees constructed using the mitochondrially encoded cytochrome b. However, an important element distinguishes the trees: the placement of Plasmodium elongatum near the base of the plastid tree, indicating an ancient lineage of parasites in birds that branches from the tree prior to other lineages of avian malaria and the human parasite, P. falciparum.

Effects of interruption of apicoplast function on malaria infection, development, and transmission

A chloroplast-like organelle is present in many species of the Apicomplexa phylum. We have previously demonstrated that the plastid organelle of Plasmodium faciparum is essential to the survival of the blood-stage malaria parasite in culture. One known function of the plastid organelle in another Apicomplexan, Toxoplasma gondii, involves the formation of the parasitophorous vacuole. The effects of interruption of plastid function on sporozoites and sexual-stage parasites have not been investigated. In our previous studies of the effects of thiostrepton, a polypeptide antibiotic from streptococcus spp., on erythrocytic schizongony of the human malaria P. falciparium, we found that this antibiotic appears to interact with the guanosine triphosphatase (GTPase) binding domain of the organellar large subunit ribosomal RNA, as it does in bacteria. We investigate here the effects of this drug on life-cycle stages of the malaria parasite in vivo. Preincubation of mature infective sporozoites with thiostrepton has no observable effect on their infectivity. Sporozoite infection both by mosquito bite and sporozoite injection was prevented by pretreatment of mice with thiostrepton. Thiostrepton eliminates infection with erythrocytic forms of Plasmodium berghei in mice. Clearance of infected red blood cells follows the delayed kinetics associated with drugs that interact with the apicoplast. Thiostrepton treatment of infected mice reduces transmission of parasites by more than ten-fold, indicating that the plastid has a role in sexual development of the parasite. These results indicate that the plastid function is accessible to drug action in vivo and important to the development of both sexual and asexual forms of the parasite.

PLASMODIUM JUXTANUCLEARE ASSOCIATED WITH MORTALITY IN BLACK-FOOTED PENGUINS (SPHENISCUS DEMERSUS) ADMITTED TO A REHABILITATION CENTER

Abstract Five black-footed penguins (Spheniscus demersus) admitted to the Southern African Foundation for the Conservation of Coastal Birds, in Cape Town, South Africa, died from malaria infection. Evidence for malaria as the cause of death included antemortem clinical signs, parasitemia, splenomegaly, pulmonary edema, and the presence of histologically visible schizonts in the reticuloendothelial system. A portion of the malarial small subunit ribosomal ribonucleic acid gene was detected by polymerase chain reaction from postmortem blood samples from all the birds. A species-specific variable region of this gene was compared with the same region on genes from other known avian malarial organisms, establishing that Plasmodium juxtanucleare was involved.

The Effects of Glucose Concentration on the Reciprocal Regulation of rRNA Promoters in Plasmodium falciparum

The developmental progression of Plasmodium falciparum is remarkably sensitive to glucose concentration. We have investigated the effects of glucose concentration on the parasite development cycle as reflected by changes of ribosomal RNA (rRNA) transcription. We showed that glucose starvation differentially affects transcriptional control of the rRNA genes by sharply repressing transcription from those loci involved with asexual development of the parasite while up-regulating transcription at those loci involved with sexual development of the parasite. Temperature change also effects regulation of transcription. We found that the effects of temperature and glucose were synergistic. We identified and compared the upstream region of the transcription start sites of each gene. These putative promoter structures are considerably different from one another and contain structures remarkably similar to rRNA control elements in other organisms.

Preliminary results of an anticircumsporozoite DNA vaccine trial for protection against avian malaria in captive African black-footed penguins (Spheniscus demersus)

Abstract Captive juvenile African black-footed penguins (Spheniscus demersus) housed in an outdoor enclosure at the Baltimore Zoo have an average 50% mortality from avian malarial (Plasmodium sp.) infection each year without intense monitoring for disease and chemotherapeutic intervention. During the 1996 malaria transmission season, the safety and efficacy of an anti-circumsporozoite (CSP) DNA vaccine encoding the Plasmodium gallinaceum CSP protein against P. relictum were studied. The goal was to reduce clinical disease and death without initiating sterile immunity after release into an area with stable, endemic avian malaria. The birds were monitored for adverse clinical signs associated with vaccination, the stimulation of an anti-CSP antibody response, and protection afforded by the vaccine. The presence of P. relictum in trapped culicine mosquitoes within the penguin enclosure was monitored to assess parasite pressure. Among the vaccinated penguins, the parasitemia rate dropped from approximately 50% to approximately 17% despite intense parasite pressure, as determined by mosquito infection rate. During the year of the vaccine trial, no mortalities due to malaria occurred and no undesirable vaccination side effects occurred. This is the first trial of an antimalarial vaccine in a captive penguin colony.

CORRELATED CHANGES IN δ‐CRYSTALLIN SYNTHESIS AND ION CONCENTRATIONS IN THE EMBRYONIC CHICK LENS: SUMMARY, CURRENT EXPERIMENTS AND SPECULATIONS

We have described a correlation between the ratio of synthesis of the higher to the lower molecular weight polypeptides of δ‐crystallin and the intracellular concentrations of Na+ and K+ in the cultured embryonic chick lens. The only structural differences we have found between the larger and smaller δ‐crystallin polypeptides are two, acidic, methionine‐containing tryptic peptides which are absent from the lower molecular weight polypeptides. We do not yet know whether the alteration in the ratio of synthesis of the polypeptides is regulated at the post‐transcriptional or post‐translational level. We are continuing to investigate this problem by studies on the cell‐free synthesis of δ‐crystallin and by analysis of the sequences and organization of δ‐crystallin DNA.

Hundreds of microsatellites for genotyping Plasmodium yoelii parasites

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.

Measuring the effects of an ever-changing environment on malaria control.

ABSTRACT The effectiveness of malaria control measures depends not only on the potency of the control measures themselves but also upon the influence of variables associated with the environment. Environmental variables have the capacity either to enhance or to impair the desired outcome. An optimal outcome in the field, which is ultimately the real goal of vaccine research, will result from prior knowledge of both the potency of the control measures and the role of environmental variables. Here we describe both the potential effectiveness of control measures and the problems associated with testing in an area of endemicity. We placed canaries with different immunologic backgrounds (e.g., naïve to malaria infection, vaccinated naïve, and immune) directly into an area where avian malaria, Plasmodium relictum, is endemic. In our study setting, canaries that are naïve to malaria infection routinely suffer approximately 50% mortality during their first period of exposure to the disease. In comparison, birds vaccinated and boosted with a DNA vaccine plasmid encoding the circumsporozoite protein of P. relictum exhibited a moderate degree of protection against natural infection (P < 0.01). In the second year we followed the fate of all surviving birds with no further manipulation. The vaccinated birds from the first year were no longer statistically distinguishable for protection against malaria from cages of naïve birds. During this period, 36% of vaccinated birds died of malaria. We postulate that the vaccine-induced protective immune responses prevented the acquisition of natural immunity similar to that concurrently acquired by birds in a neighboring cage. These results indicate that dominant environmental parameters associated with malaria deaths can be addressed before their application to a less malleable human system.