The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.

Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

Anopheles species composition explains differences in Plasmodium transmission in La Guajira, northern Colombia

Malaria in La Guajira, the most northern state of Colombia, shows two different epidemiological patterns. Malaria is endemic in the municipality of Dibulla whereas in Riohacha it is characterised by sporadic outbreaks. This study aimed to establish whether differences in transmission patterns could be attributed to different vector species. The most abundant adult female species were Anopheles aquasalis, exclusive to Riohacha, and Anopheles darlingi, restricted to Dibulla. Anopheles mosquitoes were identified using morphology and the molecular markers internal transcribed spacer 2 and cytochrome c oxidase I. All specimens (n = 1,393) were tested by ELISA to determine natural infection rates with Plasmodium falciparum and Plasmodium vivax. An. darlingi was positive for P. vivax 210, with an infection rate of 0.355% and an entomological inoculation rate of 15.87 infective bites/person/year. Anopheles albimanus larvae were the most common species in Riohacha, found in temporary swamps; in contrast, in Dibulla An. darlingi were detected mainly in permanent streams. Distinctive species composition and larval habitats in each municipality may explain the differences in Plasmodium transmission and suggest different local strategies should be used for vector control.

Structure-based drug design studies of the interactions ofent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparumsarco/endoplasmic reticulum Ca2+-ATPase PfATP6

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosawere tested in silico against the Plasmodium falciparumCa2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium bergheistrain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivaxandPlasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodium vivaxmalaria cases in China that are imported across its Southeast Asia border, especially in the China-Myanmar border area (CMB). To date, little is known about the genetic diversity of P. vivaxin this region. In this paper, we report the first genome sequencing of a P. vivaxisolate (CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto 96.43% of the P. vivaxSalvador I reference strain (Sal I) genome with 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Using the de novoassembly approach, we generated 8,541 scaffolds and assembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, we identified all 295 known virgenes, which is the largest subtelomeric multigene family in malaria parasites. These results provide an important foundation for further research onP. vivaxpopulation genetics.

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.

Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and specific real-time PCR assays

Résumé : Un nombre croissant de cas de malaria chez les voyageurs et migrants a été rapporté. Bien que l'analyse microscopique des frottis sanguins reste traditionnellement l'outil diagnostic de référence, sa fiabilité dépend considérablement de l'expertise de l'examinateur, pouvant elle-même faire défaut sous nos latitudes. Une PCR multiplex en temps réel a donc été développée en vue d'une standardisation du diagnostic. Un ensemble d'amorces génériques ciblant une région hautement conservée du gène d'ARN ribosomial 18S du genre Plasmodium a tout d'abord été conçu, dont le polymorphisme du produit d'amplification semblait suffisant pour créer quatre sondes spécifiques à l'espèce P. falciparum, P. malariae, P. vivax et P. ovale. Ces sondes utilisées en PCR en temps réel se sont révélées capables de détecter une seule copie de plasmide de P. falciparum, P. malariae, P. vivax et P. ovale spécifiquement. La même sensibilité a été obtenue avec une sonde de screening pouvant détecter les quatre espèces. Quatre-vingt-dix-sept échantillons de sang ont ensuite été testés, dont on a comparé la microscopie et la PCR en temps réel pour 66 (60 patients) d'entre eux. Ces deux méthodes ont montré une concordance globale de 86% pour la détection de plasmodia. Les résultats discordants ont été réévalués grâce à des données cliniques, une deuxième expertise microscopique et moléculaire (laboratoire de Genève et de l'Institut Suisse Tropical de Bâle), ainsi qu'à l'aide du séquençage. Cette nouvelle analyse s'est prononcé en faveur de la méthode moléculaire pour tous les neuf résultats discordants. Sur les 31 résultats positifs par les deux méthodes, la même réévaluation a pu donner raison 8 fois sur 9 à la PCR en temps réel sur le plan de l'identification de l'espèce plasmodiale. Les 31 autres échantillons ont été analysés pour le suivi de sept patients sous traitement antimalarique. Il a été observé une baisse rapide du nombre de parasites mesurée par la PCR en temps réel chez six des sept patients, baisse correspondant à la parasitémie déterminée microscopiquement. Ceci suggère ainsi le rôle potentiel de la PCR en temps réel dans le suivi thérapeutique des patients traités par antipaludéens. Abstract : There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

Expression and one-step purification of Plasmodium proteins in dictyostelium.

Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.