The impact of malaria-protective red blood cell polymorphisms on parasite biomass in children with severe Plasmodium falciparum malaria.

Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. Plasma concentrations of P. falciparum Histidine-Rich Protein 2 (PfHRP2) have diagnostic and prognostic value in severe malaria. We investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of PfHRP2 to parasite density) as quantitative traits for case-only genetic association studies of severe malaria. Data from 2198 Kenyan children diagnosed with severe malaria, genotyped for 14 major candidate genes, show that polymorphisms in four major red cell genes that lead to hemoglobin S, O blood group, α-thalassemia, and the Dantu blood group, are associated with substantially lower admission plasma PfHRP2 concentrations, consistent with protective effects against extensive parasitized erythrocyte sequestration. In contrast the known protective ATP2B4 polymorphism is associated with higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for the mechanism of protection of ATP2B4.

Forces acting on codon bias in malaria parasites.

Malaria parasite genomes have a range of codon biases, with Plasmodium falciparum one of the most AT-biased genomes known. We examined the make up of synonymous coding sites and stop codons in the core genomes of representative malaria parasites, showing first that local DNA context influences codon bias similarly across P. falciparum, P. vivax and P. berghei, with suppression of CpG dinucleotides and enhancement of CpC dinucleotides, both within and aross codons. Intense asexual phase gene expression in P. falciparum and P. berghei is associated with increased A3:G3 bias but reduced T3:C3 bias at 2-fold sites, consistent with adaptation of codons to tRNA pools and avoidance of wobble tRNA interactions that potentially slow down translation. In highly expressed genes, the A3:G3 ratio can exceed 30-fold while the T3:C3 ratio can be less than 1, according to the encoded amino acid and subsequent base. Lysine codons (AAA/G) show distinctive behaviour with substantially reduced A3:G3 bias in highly expressed genes, perhaps because of selection against frameshifting when the AAA codon is followed by another adenine. Intense expression is also associated with a strong bias towards TAA stop codons (found in 94% and 89% of highly expressed P. falciparum and P. berghei genes respectively) and a proportional rise in the TAAA stop 'tetranucleotide'. The presence of these expression-linked effects in the relatively AT-rich malaria parasite species adds weight to the suggestion that AT-richness in the Plasmodium genus might be a fitness adaptation. Potential explanations for the relative lack of codon bias in P. vivax include the distinct features of its lifecycle and its effective population size over evolutionary time.

Field-based flow cytometry for ex vivo characterization of Plasmodium vivax and P. falciparum antimalarial sensitivity.

Ex vivo antimalarial sensitivity testing in human malaria parasites has largely depended on microscopic determination of schizont maturation. While this microscopic method is sensitive, it suffers from poor precision and is laborious. The recent development of portable, low-cost cytometers has allowed us to develop and validate a simple, field-optimized protocol using SYBR green and dihydroethidium for the accurate and objective determination of antimalarial drug sensitivity in freshly isolated Plasmodium vivax and Plasmodium falciparum.

Antimalarial drugs: recent advances in molecular determinants of resistance and their clinical significance.

Molecular determinants of antimalarial drug resistance are useful and informative tools that complement phenotypic assays for drug resistance. They also guide the design of strategies to circumvent such resistance once it has reached levels of clinical significance. Established resistance to arylaminoalcohols such as mefloquine and lumefantrine in SE Asia is mediated primarily by gene amplification of the P. falciparum drug transporter, pfmdr1. Single nucleotide polymorphisms in pfmdr1, whether assessed in field isolates or transfection experiments, are associated with changes in IC(50) values (to arylaminoalcohols and chloroquine), but not of such magnitude as to influence clinical treatment outcomes. Recently described emerging in vitro resistance to artemisinins in certain areas correlates with mutations in the SERCA-like sequence PfATP6 and supports PfATP6 as a key target for artemisinins.

Artemisinins.

Artemisinins were discovered to be highly effective antimalarial drugs shortly after the isolation of the parent artemisinin in 1971 in China. These compounds combine potent, rapid antimalarial activity with a wide therapeutic index and an absence of clinically important resistance. Artemisinin containing regimens meet the urgent need to find effective treatments for multidrug resistant malaria and have recently been advocated for widespread deployment. Comparative trials of artesunate and quinine for severe malaria are in progress to see if the persistently high mortality of this condition can be reduced.

Occludin expression in microvessels of neoplastic and non-neoplastic human brain.

The tight junction protein occludin 'glues' normal, adjacent brain microvessel endothelial cells together. Malignant brain tumours cause cerebral oedema because they have leaky endothelial tight junctions, which allow plasma fluid to enter the brain from the microvessel lumen. In order to identify molecular abnormalities in tumour endothelial tight junctions, we investigated occludin expression in microvessels from adult human non-neoplastic brain tissue using immunohistochemistry and immunoblotting. The proportions of microvessels immunolabelling for occludin were >2/3 in 5/5 non-neoplastic brain tissue samples, >1/3 in 5/5 low grade (Daumas-Duport I or II) astrocytomas and <1/3 in 5/5 high grade (III or IV) astrocytomas and 6/6 metastatic adenocarcinomas. Six non-neoplastic brain tissue immunoblots gave a 55-kDa occludin band, three low-grade astrocytomas gave 55-kDa and 60-kDa bands, 13 high-grade astrocytomas gave 60-kDa or no band and four adenocarcinomas did not give an occludin band. Expression of 55-kDa occludin inversely correlated with the presence of contrast enhancement on computed tomograms (P < 0.001). Electron microscopy showed open endothelial tight junctions in 0/2 non-neoplastic human brain specimens and 2/2 high-grade astrocytomas. We suggest that loss of 55-kDa occludin expression in human brain tumours may contribute to endothelial tight junction opening. Characterizing the molecular pathology of brain endothelial tight junctions may facilitate the design of novel drugs against cerebral oedema.

Characterization of P-type ATPase 3 in Plasmodium falciparum.

We report the nucleotide sequence, derived amino acid sequence and expression profile of P-type ATPase 3 (PfATPase3) from Plasmodium falciparum. An open reading frame of 7362 nucleotides, interrupted by a single intron of 168 nt, encoded a protein product of 2394 amino acids with a predicted MW of 282791 Da. Hydropathy analysis of PfATPase3 revealed six amino-terminal and six carboxyl-terminal membrane spanning regions (M1-12) flanking a large hydrophilic domain with a smaller hydrophilic loop between M4 and M5. Based on a phylogenetic comparison of conserved domains present in P-type ATPases from other organisms, PfATPase3 resembled a Type-V ATPase for which the transport affinity is unknown. The PfATPase3 topology was interrupted by four regions, termed 'inserts', unique to malarial P-type ATPases, which were high in asparagine residues and charged amino acids (inserts I1-I4). Inserts I1 and I3 also contained repeated amino acid motifs. The number and composition of repeated amino acid motifs in insert I3 were variable in seven P. falciparum strains tested. PfATPase3 was 80.2% similar to the non-insert portions of P. yoelii ATPase3, although their inserts differed in length and composition. PfATPase3 mRNA was most abundant relative to beta-tubulin during the latter half of the erythrocytic cycle and was also present in gametocytes. Using affinity-purified antibody to a 14 amino acid PfATPase3 epitope, a 260 kDa protein was detected by Western analysis. Based on immunofluorescence, the PfATPase3 protein was located intracellularly in gametocytes and, to a lesser extent, in late erythrocytic stages.

Hexose transport in asexual stages of Plasmodium falciparum and kinetoplastidae.

The hexose sugar, glucose, is a vital energy source for most organisms and an essential nutrient for asexual stages of Plasmodium falciparum. Kinetoplastid organisms (e.g. Trypanosoma and Leishmania spp) also require glucose at certain critical stages of their life cycles. Although phylogenetically unrelated, these organisms share many common challenges during the mammalian stages of a parasitic life cycle, and possess hexose uptake mechanisms that are amenable to study using similar methods. Defining hexose permeation pathways into parasites might expose an Achilles' heel at which both antidisease and antiparasite measures can be aimed. Understanding the mode of entry of glucose also presents a good general model for substrate acquisition in multicompartment systems. In this review, Sanjeev Krishna and colleagues summarize current understanding of hexose transport processes in P. falciparum and provide a comparison with data obtained from kinetoplastids.

Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in Xenopus oocytes of the P. falciparum asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter, K(m) approximately 1.0 mM) also transports fructose (K(m) approximately 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured P. falciparum, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.

Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.

Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter of P. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (Km = 0.48 mM) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition.

Recent developments in the management of malaria.

The last decade has seen in increase in our understanding of the pathophysiology of severe malaria in both adults and children. However, this increased knowledge has yet to be matched by progress in the clinical management of this medical emergency. In the last few years a wide variety of measures have been advocated to lessen the mortality due to severe malaria, but none have shown a significant benefit in terms of mortality or morbidity. In this review we concentrate on aspects of the pathophysiology of malaria which are amenable to intervention at present or in the near future. In the field of uncomplicated malaria problems remain mostly related to the continued spread of drug-resistance and the limited array of available drugs. Outlined are a number of important advances in antimalarial pharmacology and parasite biology that may lead to future improvement in the care of patients with malaria.

Expression of parasite transporters in Xenopus oocytes.

The Xenopus laevis oocyte heterologous expression system is particularly useful for the study of transporter proteins. We demonstrated the utility of this expression system for studies on Plasmodium falciparum transporters by inducing increased uptake of metabolites or their analogues (nucleosides, nucleobases, lactate and glucose) into oocytes after microinjection of mRNA obtained from asexual stages of P. falciparum. We identified a hexose transporter of P. falciparum (PfHT1) and studied its function. Higher levels of functional activity are obtained when 5' and 3' untranslated Xenopus globin gene sequences and a strong Kozak consensus are included in RNA used for microinjection studies. PfHT1 is a saturable, sodium-independent and stereospecific transporter with a relatively high affinity for glucose (K(m) = 0.48 mM). Competition experiments with glucose analogues show that hydroxyl groups at positions C3 and C4 are important for ligand binding. mRNA levels for PfHT1 are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. When hypoglycaemia complicates cerebral malaria, modelling studies using data obtained from oocyte experiments suggest that the high affinity of PfHT1 may increase the proportion of glucose taken up by parasites compared with that transported across the blood-brain barrier.

Expression of substrate-specific transporters encoded by Plasmodium falciparum in Xenopus laevis oocytes.

When the malarial parasite Plasmodium falciparum multiplies in erythrocytes it dramatically increases uptake of essential metabolic precursors (nucleosides, nucleobases and glucose) and export of lactic acid by undefined mechanisms. The first evidence is provided here, by a detailed study in Xenopus laevis oocytes, that several specific nutrient transporters are the product of P. falciparum genes. We report the expression of nucleoside, nucleobase, hexose and monocarboxylate transport systems in Xenopus oocytes when injected with mRNA isolated from asexual stages of developing P. falciparum parasites. Their properties are distinct from transport events occurring at the infected erythrocyte membrane or the electrophysiologically identified channel localised to the parasitophorous vacuolar membrane. These novel transporters are substrate-specific and stereoselective, and represent a key regulatory step in the acquisition and export of metabolites by intraerythrocytic P. falciparum.

Assessment of cochlear damage after pneumococcal meningitis using otoacoustic emissions.

Three cases are reported of children in Ghana with pneumococcal meningitis and differing degrees of hearing loss. The children were examined up to 12 d after admission by means of otoacoustic emissions. The technique is objective, non-invasive, quick (< 5 min per ear) and suitable for use in paediatric wards.