Comparative genomics of the neglected human malaria parasite Plasmodium vivax

The human malaria parasite Plasmodium vivax is responsible for 25 - 40% of the similar to 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non- human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax

The population structure of Plasmodium vivax remains elusive. The markers of choice for large-scale population genetic studies of eukaryotes, short tandem repeats known as microsatellites, have been recently reported to be less polymorphic in R vivax. Here we investigate the microsatellite diversity and geographic structure in P vivax, at both local and global levels, using 14 new markers consisting of tri- or tetranucleotide repeats. The local-level analysis, which involved 50 field isolates from Sri Lanka, revealed unexpectedly high diversity (average virtual heterozygosity [H-E], 0.807) and significant multilocus linkage disequilibrium in this region of low malaria endemicity. Multiple-clone infections occurred in 60% of isolates sampled in 2005. The global-level analysis of field isolates or monkey-adapted strains identified 150 unique haplotypes among 164 parasites from four continents. Individual P. vivax isolates could not be unambiguously assigned to geographic populations. For example, we found relatively low divergence among parasites from Central America, Africa, Southeast Asia and Oceania, but substantial differentiation between parasites from the same continent (South Asia and Southeast Asia) or even from the same country (Brazil). Parasite relapses, which may extend the duration of P. vivax carriage in humans, are suggested to facilitate the spread of strains across continents, breaking down any pre-existing geographic structure. (C) 2008 Elsevier B.V. All rights reserved.

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Habitat suitability of Anopheles vector species and association with human malaria in the Atlantic Forest in south-eastern brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vitamin B6-Dependent Enzymes in the Human Malaria Parasite Plasmodium falciparum: A Druggable Target?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background: The mechanisms by which humans regulate pro-and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. Methodology/Principal Findings: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-alpha receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-alpha, IL-10/interferon (IFN)-gamma, IL-10/IL-6 and sTNFRII/TNF-alpha ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-alpha receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. Conclusions: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.

Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen (1O2). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of 1O2 by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While 1O2 is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of 1O2 we have established a new cytometric method that allows the stage specific quantification of 1O2. Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200600 pixels for rings, 7001,200 pixels for trophozoites and 1,4002,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous 1O2 of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that 1O2 derives predominantly from the digestion of hemoglobin. (c) 2012 International Society for Advancement of Cytometry

Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

FULL-malaria: a database for a full-length enriched cDNA library from human malaria parasite, Plasmodium falciparum

FULL-malaria is a database for a full-length-enriched cDNA library from the human malaria parasite Plasmodium falciparum (http://133.11.149.55/). Because of its medical importance, this organism is the first target for genome sequencing of a eukaryotic pathogen; the sequences of two of its 14 chromosomes have already been determined. However, for the full exploitation of this rapidly accumulating information, correct identification of the genes and study of their expression are essential. Using the oligo-capping method, we have produced a full-length-enriched cDNA library from erythrocytic stage parasites and performed one-pass reading. The database consists of nucleotide sequences of 2490 random clones that include 390 (16%) known malaria genes according to BLASTN analysis of the nr-nt database in GenBank; these represent 98 genes, and the clones for 48 of these genes contain the complete protein-coding sequence (49%). On the other hand, comparisons with the complete chromosome 2 sequence revealed that 35 of 210 predicted genes are expressed, and in addition led to detection of three new gene candidates that were not previously known. In total, 19 of these 38 clones (50%) were full-length. From these obser­vations, it is expected that the database contains ∼1000 genes, including 500 full-length clones. It should be an invaluable resource for the development of vaccines and novel drugs.

Immunoregulation in human malaria: the challenge of understanding asymptomatic infection

Asymptomatic Plasmodium infection carriers represent a major threat to malaria control worldwide as they are silent natural reservoirs and do not seek medical care. There are no standard criteria for asymptomatic Plasmodium infection; therefore, its diagnosis relies on the presence of the parasite during a specific period of symptomless infection. The antiparasitic immune response can result in reduced Plasmodium sp. load with control of disease manifestations, which leads to asymptomatic infection. Both the innate and adaptive immune responses seem to play major roles in asymptomatic Plasmodium infection; T regulatory cell activity (through the production of interleukin- 10 and transforming growth factor-β) and B-cells (with a broad antibody response) both play prominent roles. Furthermore, molecules involved in the haem detoxification pathway (such as haptoglobin and haeme oxygenase-1) and iron metabolism (ferritin and activated c-Jun N-terminal kinase) have emerged in recent years as potential biomarkers and thus are helping to unravel the immune response underlying asymptomatic Plasmodium infection. The acquisition of large data sets and the use of robust statistical tools, including network analysis, associated with welldesigned malaria studies will likely help elucidate the immune mechanisms responsible for asymptomatic infection.

Genetic polymorphisms in TLR4, CR1 and Duffy genes are not associated with malaria resistance in patients from Baixo Amazonas region, Brazil

The main purpose of this research was to analyze the relation of the genetic polymorphisms frequently expressed by antigen-presenting cells, erythrocytes and malaria susceptibility/resistance with the human malaria infection cases. The sample used consisted of 23 Plasmodium vivax ( Pv)- and P. falciparum ( Pf)-infected patients, and 21 healthy individuals as a control group, from the Baixo Amazonas population in Para, Brazil. The Asp299Gly polymorphisms in the Toll-like receptor 4 ( TLR4), and Gly42Asp, Arg89Cys, Ala100Thr, and T-33C in the Duffy gene ( FY) were analyzed by restriction fragment length polymorphism-polymerase chain reaction. The Lys1590Glu and Arg1601Gly polymorphisms in the complement receptor type 1 (CR1) were analyzed by DNA sequencing. According to the results obtained and statistical analysis considering a significance level or alpha = 0.01, we conclude that the low heterozygote frequency (2.27%) for the Asp299Gly mutation, detected in the TLR4 gene, is not related to the Pv and Pf infections in the patients analyzed. Also, the promoter region GATA-1 analysis of the FY gene in the Pv-infected patients showed that the heterozygote frequency for the T-33C mutation (11.36% of the infected patients and 20.45% of the control patients) is not related to infection resistance. Regarding the CR1 gene, the observed heterozygote frequency (9.09%) for the Arg1601Gly mutation in Pf-infected patients when compared to heterozygote frequency in the control group (18.18%) suggests that there is no correlation with infection resistance.

Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies

Background: The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized. Results: We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for similar to 40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance. Conclusion: These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.

Genome-Wide Detection of Serpentine Receptor-Like Proteins in Malaria Parasites

Serpentine receptors comprise a large family of membrane receptors distributed over diverse organisms, such as bacteria, fungi, plants and all metazoans. However, the presence of serpentine receptors in protozoan parasites is largely unknown so far. In the present study we performed a genome-wide search for proteins containing seven transmembrane domains (7TM) in the human malaria parasite Plasmodium falciparum and identified four serpentine receptor-like proteins. These proteins, denoted PfSR1, PfSR10, PfSR12 and PfSR25, show membrane topologies that resemble those exhibited by members belonging to different families of serpentine receptors. Expression of the pfsrs genes was detected by Real Time PCR in P. falciparum intraerythrocytic stages, indicating that they potentially code for functional proteins. We also found corresponding homologues for the PfSRs in five other Plasmodium species, two primate and three rodent parasites. PfSR10 and 25 are the most conserved receptors among the different species, while PfSR1 and 12 are more divergent. Interestingly, we found that PfSR10 and PfSR12 possess similarity to orphan serpentine receptors of other organisms. The identification of potential parasite membrane receptors raises a new perspective for essential aspects of malaria parasite host cell infection.