Sialic acids: Key determinants for invasion by the Apicomplexa

Sialic acids are ubiquitously found on the surface of all vertebrate cells at the extremities of glycan chains and widely exploited by viruses and bacteria to enter host cells. Carbohydrate-bearing receptors are equally important for host cell invasion by the obligate intracellular protozoan parasites of the phylum Apicomplexa. Host cell entry is an active process relying crucially on proteins that engage with receptors on the host cell surface and promote adhesion and internalisation. Assembly into complexes, proteolytic processing and oligomerization are important requirements for the functionality of these adhesins. The combination of adhesive proteins with varying stringency in specificity confers some flexibility to the parasite in face of receptor heterogeneity and immune pressure. Sialic acids are now recognised to critically contribute to selective host cell recognition by various species of the phylum.     

Cellular architecture of Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum is a protozoan parasite that is responsible for the most pathogenic form of human malaria. The particular virulence of this parasite derives from its ability to develop within the erythrocytes of its host and to subvert their function. The intraerythrocytic parasite devours haemoglobin, and remodels its host cell to cause adhesion to blood vessel walls. Ultrastructural studies of P. falciparum have played a major role in defining its cell architecture and in resolving cell biology controversies. Here we review some of the early studies and describe some recent developments in electron microscopy techniques that have revealed information about the organization of the parasite in the blood stage of development. We present images of P. falciparum at different stages of the life cycle and highlight some of the plasmodium-specific organelles, the haemoglobin digestive apparatus and the membrane structures that are elaborated in the host cell cytoplasm to traffic virulence proteins to the erythrocyte surface. We describe methods for whole cell ultrastructural imaging that can provide three-dimensional views of intraerythrocytic development.     

Plasmodium vivax: Comparison of the immune responses between oral and parenteral immunization of rPv54 in BALB/c mice

The merozoite surface protein-1 (MSP-1) from Plasmodium vivax was evaluated as an oral vaccine candidate by cloning and expressing the interspecies conserved block 10 (ICB10) of the MSP-1 from a Korean isolate in Escherichia coli. The expressed fusion protein contained ICB10 and a maltose-binding protein (MBP), rPv54, has a molecular weight of approximately 54 kDa as determined by SDS-PAGE analysis. IgG against rPv54 was successfully produced in BALB/c mice by oral immunization and sustained for more than 4 months. IgG2b was dominantly produced in both oral and parenteral immunizations. The rPv54 increased the frequency of NK, NKT, CD4+ T, CD8+ T, and B cells in both immunizations. IL-5 and TNF-α were increased in both significantly. In conclusion, rPv54 might be a valuable potential vaccine candidate for the oral and parenteral immunization against vivax malaria.     

Plasmodium yoelii: Influence of immune modulators on the development of the liver stage

Plasmodium sporozoites suppress the respiratory burst and antigen presentation of Kupffer cells, which are regarded as the portal of invasion into hepatocytes. It is not known whether immune modulation of Kupffer cells can affect the liver stage. In the present study, we found that sporozoites inoculated into Wistar rats could be detected in the liver, spleen, and lung; however, most sporozoites were arrested in the liver. Sporozoites were captured by Kupffer cells lined with endothelial cells in the liver sinusoid before hepatocyte invasion. Pretreatment with TLR3 agonist poly(I:C) and TLR2 agonist BCG primarily activated Kupffer cells, inhibiting the sporozoite development into the exoerythrocytic form, whereas Kupffer cell antagonists dexamethasone and cyclophosphamide promoted development of the liver stage. Our data suggests that sporozoite development into its exoerythrocytic form may be associated with Kupffer cell functional status. Immune modulation of Kupffer cells could be a promising strategy to prevent malaria parasite infection.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

Inhibitory effect of ursolic acid derivatives on hydrogen peroxide- and glutathione-mediated degradation of hemin: A possible additional mechanism of action for antimalarial activity

Compounds obtained by the condensation of ursolic acid (UA) with 1,4-bis(3-aminopropyl)piperazines have previously been shown as cytocidal to Plasmodium falciparum strains. Preliminary results indicated that the inhibition of β-hematin formation (one of the possible mechanisms of action of antimalarial drugs) was achieved by a few of these molecules with varying efficiencies. To gain further insight in the antimalarial action of UA derivatives, we report here the results of additional pathways that may explain their in vitro cytocidal activity such as inhibition of hemin degradation by H₂O₂ or glutathione (GSH). H₂O₂-mediated hemin degradation was drastically reduced by hydroxybenzyl-substituted UA derivatives while UA and intermediate compounds displayed weaker inhibitory actions. The results of GSH-mediated hemin degradation inhibition did not parallel those of H₂O₂ degradation as hydroxybenzyl-substituted UA only proved to be a weak inhibitor. As H₂O₂ interaction with the iron moiety of hemin is the first step towards its degradation, we assume that the interaction of our products with the ferric ion in the hemin structure is of upmost importance in inhibiting its peroxidative degradation. A two-step mechanism of action implying (1) stacking of the acetylursolic acid structure to hemin and (2) additive protection of hemin ferric iron from H₂O₂ by hydroxyphenyl groups through steric hindrance and/or trapping of oxygen reactive species in the direct neighborhood of ferric iron can be put forward. For GSH degradation pathway, grafting of UA structure with a piperazine structure gave the best inhibition, pleading for the implication of this latter moiety in the inhibitory process.     

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.     

Role of Plasmodium berghei cGMP-dependent Protein Kinase in Late Liver Stage Development

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.     

Characterization of anti-var2CSA-PfEMP1 cytoadhesion inhibitory mouse monoclonal antibodies

Pregnancy-associated malaria (PAM) is associated with the massive sequestration of erythrocytes infected with CSA-binding parasites in the placenta. Natural protective immunity against PAM is acquired during the course of pregnancies, with the development of anti-PfEMP1 antibodies recognizing placental infected erythrocytes (IEs) from different geographical regions. Mouse monoclonal antibodies (mabs) were raised against Plasmodium falciparum variant surface proteins expressed by CSA-binding parasites. These mabs blocked 0-60% of CSA-binding parasite adhesion and immunoprecipitated a 350 kDa 125I-labeled PfEMP1(CSA). Two var2CSA domains expressed on the surface of CHO cells (DBL5epsilon and DBL6epsilon) were identified as the targets of three of four antibodies inhibiting CSA binding. Two of these antibodies also recognized either DBL2x or DBL3x, suggesting that some epitopes may be common to several var2CSA domains. These mabs also specifically selected CSA-binding IEs and facilitated the purification from IE extracts of the native var2CSA ligand. This purified ligand elicited antibodies in immunized mice inhibiting efficiently IE(CSA) cytoadhesion. Based on our findings, we provide the first demonstration that the parasite var2CSA surface protein can elicit inhibitory antibodies and define here the subunits of the var2CSA ligand suitable for use in vaccine development.     

Five labdane diterpenoids from the seeds of Aframomum zambesiacum

Five labdane diterpenoids, (3-5), zambesiacolactone A (7) and zambesiacolactone B (8), were isolated from the seeds of Aframomum zambesiacum (Baker) K. Schum., along with five known labdanes and a linear sesquiterpene, nerolidol. Their structures were elucidated by spectroscopic analysis. Their antiplasmodial activity was evaluated in vitro against Plasmodium falciparum. Compound 3 was the most active with an IC(50) value of 4.97 microM.