Investigating the Pathogenesis of Severe Malaria: A Multidisciplinary and Cross-Geographical Approach.

More than a century after the discovery of Plasmodium spp. parasites, the pathogenesis of severe malaria is still not well understood. The majority of malaria cases are caused by Plasmodium falciparum and Plasmodium vivax, which differ in virulence, red blood cell tropism, cytoadhesion of infected erythrocytes, and dormant liver hypnozoite stages. Cerebral malaria coma is one of the most severe manifestations of P. falciparum infection. Insights into its complex pathophysiology are emerging through a combination of autopsy, neuroimaging, parasite binding, and endothelial characterizations. Nevertheless, important questions remain regarding why some patients develop life-threatening conditions while the majority of P. falciparum-infected individuals do not, and why clinical presentations differ between children and adults. For P. vivax, there is renewed recognition of severe malaria, but an understanding of the factors influencing disease severity is limited and remains an important research topic. Shedding light on the underlying disease mechanisms will be necessary to implement effective diagnostic tools for identifying and classifying severe malaria syndromes and developing new therapeutic approaches for severe disease. This review highlights progress and outstanding questions in severe malaria pathophysiology and summarizes key areas of pathogenesis research within the International Centers of Excellence for Malaria Research program

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy

Regulatory Hotspots in the Malaria Parasite Genome Dictate Transcriptional Variation

The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs) in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant expression quantitative trait locus. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to an increased copy number of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites

A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites

Kinetics and Ligand-Binding Preferences of Mycobacterium tuberculosis Thymidylate Synthases, ThyA and ThyX

Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.After cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k(cat) values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Thus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis

Microfluidic modeling of cell-cell interactions in malaria pathogenesis.

The clinical outcomes of human infections by Plasmodium falciparum remain highly unpredictable. A complete understanding of the complex interactions between host cells and the parasite will require in vitro experimental models that simultaneously capture diverse host-parasite interactions relevant to pathogenesis. Here we show that advanced microfluidic devices concurrently model (a) adhesion of infected red blood cells to host cell ligands, (b) rheological responses to changing dimensions of capillaries with shapes and sizes similar to small blood vessels, and (c) phagocytosis of infected erythrocytes by macrophages. All of this is accomplished under physiologically relevant flow conditions for up to 20 h. Using select examples, we demonstrate how this enabling technology can be applied in novel, integrated ways to dissect interactions between host cell ligands and parasitized erythrocytes in synthetic capillaries. The devices are cheap and portable and require small sample volumes; thus, they have the potential to be widely used in research laboratories and at field sites with access to fresh patient samples