Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes

The uptake and expression of extracellular DNA has been established as a mechanism for horizontal transfer of genes between bacterial species. Such transfer can support acquisition of advantageous elements, including determinants that affect the interactions between infectious organisms and their hosts. Here we show that erythrocyte-stage Plasmodium falciparum malaria parasites spontaneously take up DNA from the host cell cytoplasm into their nuclei. We have exploited this finding to produce levels of reporter expression in P.falciparum that are substantially improved over those obtained by electroporation protocols currently used to transfect malaria parasites. Parasites were transformed to a drug-resistant state when placed into cell culture with erythrocytes containing a plasmid encoding the human dihydrofolate reductase sequence. The findings reported here suggest that the malaria genome may be continually exposed to exogenous DNA from residual nuclear material in host erythrocytes.

A role for CD36 in the regulation of dendritic cell function

Dendritic cells (DC) are crucial for the induction of immune responses and thus an inviting target for modulation by pathogens. We have previously shown that Plasmodium falciparum-infected erythrocytes inhibit the maturation of DCs. Intact P. falciparum-infected erythrocytes can bind directly to CD36 and indirectly to CD51. It is striking that these receptors, at least in part, also mediate the phagocytosis of apoptotic cells. Here we show that antibodies against CD36 or CD51, as well as exposure to early apoptotic cells, profoundly modulate DC maturation and function in response to inflammatory signals. Although modulated DCs still secrete tumor necrosis factor-α, they fail to activate T cells and now secrete IL-10. We therefore propose that intact P. falciparum-infected erythrocytes and apoptotic cells engage similar pathways regulating DC function. These findings may have important consequences for the treatment of malaria and may suggest strategies for modulating pathological immune responses in autoimmune diseases.

Applications of pox virus vectors to vaccination: an update.

Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies.

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.

Sphingolipid synthesis as a target for chemotherapy against malaria parasites.

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.

Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy

Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10(4) P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei At doses of 10(5) and 10(6) IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6-EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA.

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

GFP-targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites.

BACKGROUND INFORMATION The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo-erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. RESULTS In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo-erythrocytic schizogony in vitro, leading to impaired parasite maturation. CONCLUSIONS Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red-blood-cell-infective merozoites.

Signalling in malaria parasites. The MALSIG consortium

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Can primaquine therapy for vivax malaria be improved?

The incidence and range of endemic malaria caused by Plasmodium vivax has expanded during the past 30 years. This parasite forms hypnozoites in the liver, creating a persistent reservoir of infection. Primaquine (PQ), introduced 50 years ago, is the only drug available to eliminate hypnozoites. However, lengthy treatment courses and follow-up periods are not conducive to assessing the effectiveness of this drug in preventing relapses. Resistance to standard therapy could be widespread. Studies are urgently needed to gauge this problem and to determine the safety, tolerability and efficacy of shorter courses and higher doses of PQ.