Gerhard Wunderlich

On the Cytoadhesion of Plasmodium vivax–Infected Erythrocytes

BACKGROUND Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Antigenic diversity and immune evasion by malaria parasites.

Malaria parasites are major human pathogens annually associated with 300 million to 500 million clinical cases worldwide and 0.5 million to 3 million deaths, mostly among children under the age of 5 years living in sub-Saharan Africa ([50][1]). Human malaria is caused by four species of parasitic

PLASMODIUM IN THE POSTGENOMIC ERA : NEW INSIGHTS INTO THE MOLECULAR CELL BIOLOGY OF MALARIA PARASITES

In this review, we bring together some of the approaches toward understanding the cellular and molecular biology of Plasmodium species and their interaction with their host red blood cells. Considerable impetus has come from the development of new methods of molecular genetics and bioinformatics, and it is important to evaluate the wealth of these novel data in the context of basic cell biology. We describe how these approaches are gaining valuable insights into the parasite-host cell interaction, including (1) the multistep process of red blood cell invasion by the merozoite; (2) the mechanisms by which the intracellular parasite feeds on the red blood cell and exports parasite proteins to modify its cytoadherent properties; (3) the modulation of the cell cycle by sensing the environmental tryptophan-related molecules; (4) the mechanism used to survive in a low Ca(2+) concentration inside red blood cells; (5) the activation of signal transduction machinery and the regulation of intracellular calcium; (6) transfection technology; and (7) transcriptional regulation and genome-wide mRNA studies in Plasmodium falciparum.

Carotenoid Biosynthesis in Intraerythrocytic Stages of Plasmodium falciparum

Carotenoids are widespread lipophilic pigments synthesized by all photosynthetic organisms and some nonphotosynthetic fungi and bacteria. All carotenoids are derived from the C40 isoprenoid precursor geranylgeranyl pyrophosphate, and their chemical and physical properties are associated with light absorption, free radical scavenging, and antioxidant activity. Carotenoids are generally synthesized in well defined subcellular organelles, the plastids, which are also present in the phylum Apicomplexa, which comprises a number of important human parasites, such as Plasmodium and Toxoplasma. Recently, it was demonstrated that Toxoplasma gondii synthesizes abscisic acid. We therefore asked if Plasmodium falciparum is also capable of synthesizing carotenoids. Herein, biochemical findings demonstrated the presence of carotenoid biosynthesis in the intraerythrocytic stages of the apicomplexan parasite P. falciparum. Using metabolic labeling with radioisotopes, in vitro inhibition tests with norflurazon, a specific inhibitor of plant carotenoid biosynthesis, the results showed that intraerythrocytic stages of P. falciparum synthesize carotenoid compounds. A plasmodial enzyme that presented phytoene synthase activity was also identified and characterized. These findings not only contribute to the current understanding of P. falciparum evolution but shed light on a pathway that could serve as a chemotherapeutic target.

Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo

ABSTRACT Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Identification, molecular cloning and functional characterization of an octaprenyl pyrophosphate synthase in intra-erythrocytic stages of Plasmodium falciparum

Isoprenoids play important roles in all living organisms as components of structural cholesterol, steroid hormones in mammals, carotenoids in plants, and ubiquinones. Significant differences occur in the length of the isoprenic side chains of ubiquinone between different organisms, suggesting that different enzymes are involved in the synthesis of these side chains. Whereas in Plasmodium falciparum the isoprenic side chains of ubiquinone contain 7-9 isoprenic units, 10-unit side chains are found in humans. In a search for the P. falciparum enzyme responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, we cloned and expressed a putative polyprenyl synthase. Polyclonal antibodies raised against the corresponding recombinant protein confirmed the presence of the native protein in trophozoite and schizont stages of P. falciparum. The recombinant protein, as well as P. falciparum extracts, showed an octaprenyl pyrophosphate synthase activity, with the formation of a polyisoprenoid with eight isoprenic units, as detected by reverse-phase HPLC and reverse-phase TLC, and confirmed by electrospray ionization and tandem MS analysis. The recombinant and native versions of the enzyme had similar Michaelis constants with the substrates isopentenyl pyrophosphate and farnesyl pyrophosphate. The recombinant enzyme could be competitively inhibited in the presence of the terpene nerolidol. This is the first report that directly demonstrates an octaprenyl pyrophosphate synthase activity in parasitic protozoa. Given the rather low similarity of the P. falciparum enzyme to its human counterpart, decaprenyl pyrophosphate synthase, we suggest that the identified enzyme and its recombinant version could be exploited in the screening of novel drugs.

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.

Antigenic Polymorphism and Naturally Acquired Antibodies to Plasmodium vivax Merozoite Surface Protein 1 in Rural Amazonians

ABSTRACT Merozoite surface protein 1 of Plasmodium vivax (PvMSP-1), a major target for malaria vaccine development, contains six highly polymorphic domains interspersed with conserved sequences. Although there is evidence that the sequence divergence in PvMSP-1 has been maintained over 5 million years by balanced selection exerted by the hosts acquired immunity, the variant specificity of naturally acquired antibodies to PvMSP-1 remains poorly investigated. Here, we show that 15 recombinant proteins corresponding to PvMSP-1 variants commonly found in local parasites were poorly recognized by 376 noninfected subjects aged 5 to 90 years exposed to malaria in rural Amazonia; less than one-third of them had detectable immunoglobulin G (IgG) antibodies to at least one variant of blocks 2, 6, and 10 that were expressed, although 54.3% recognized the invariant 19-kDa C-terminal domain PvMSP-119. Although the proportion of responders to PvMSP-1 variants increased substantially during subsequent acute P. vivax infections, the specificity of IgG antibodies did not necessarily match the PvMSP-1 variant(s) found in infecting parasites. We discuss the relative contribution of antigenic polymorphism, poor immunogenicity, and original antigenic sin (the skew in the specificity of antibodies elicited by exposure to new antigenic variants due to preexisting variant-specific responses) to the observed patterns of antibody recognition of PvMSP-1. We suggest that antibody responses to the repertoire of variable domains of PvMSP-1 to which subjects are continuously exposed are elicited only after several repeated infections and may require frequent boosting, with clear implications for the development of PvMSP-1-based subunit vaccines.

Detection of etiological agents of malaria in howler monkeys from Atlantic Forests, rescued in regions of São Paulo city, Brazil

Background  In some states of the Brazilian extra‐Amazonian region, such as the Atlantic Forest area, autochthonous human cases of malaria were related to simian malarias and vice versa.

Antigenic properties of the merozoite surface protein 1 gene of Plasmodium vivax.

Plasmodium vivax is responsible for an approximate 35 million yearly human cases of malaria. Unfortunately, due to the low mortality rate associated with it and the difficulties of continuously in vitro culturing of this parasite, vaccine development against this human malaria has been largely neglected. In here, the antigenic properties of the merozoite surface protein 1 gene of P. vivax (PvMSP-1), were studied. Thus, seven recombinant bacterial plasmids coding different regions of the PvMSP-1 protein were constructed and used to immunize BALB/c mice. The results demonstrated that a plasmid encoding the entire N-terminus comprising 682 amino acids and a plasmid encoding the C-terminus including the two juxtaposed epidermal growth factor (EGF)-like domains fused to the Hepatitis B surface antigen, were antigenic. Moreover, the elicited immune responses were similar to those reported for these same PvMSP-1 regions in natural human infections.