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.

Comparison of bioassay and high performance liquid chromatographic assay of artesunate and dihydroartemisinin in plasma

The study was a comparison of bioassay and HPLC analysis of artesunate (ARTS) and dihydroartemisinin (DHA) in plasma. ARTS and DHA in plasma samples from patients treated with ARTS were quantified by HPLC and expressed as DHA. DHA-equivalents in the same plasma samples were measured using a standardised parasite culture technique. DHA concentrations estimated by both methods were highly correlated (bioassay = 0.96 x HPLC + 11.0; r(2) = 0.92). At high concentrations ( > 12 000 nmol/l) bioassay sometimes overestimated DHA. Bioassay of active drug in plasma correlates well with specific chemical analysis by HPLC. ARTS and DHA appear to account for the total antimalarial activity in plasma after ARTS administration. (C) 2003 Elsevier Science B.V. All rights reserved.

Simple In Vitro Assay for Determining the Sensitivity of Plasmodium vivax Isolates from Fresh Human Blood to Antimalarials in Areas where P. vivax Is Endemic

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P.falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

Plasma concentrations of tafenoquine, a new long-acting antimalarial agent, in Thai soldiers receiving monthly prophylaxis

We measured plasma tafenoquine concentrations in Thai soldiers given a monthly regimen of tafenoquine to determine whether these concentrations adequately suppressed malarial infections on the Thai- Cambodian border. After receiving a treatment course of artesunate and doxycycline, 104 male soldiers were administered a loading dose of tafenoquine ( 400 mg daily for 3 days), followed by tafenoquine monthly ( 400 mg every 4 weeks) for 5 months. Consecutive monthly mean ( +/- standard deviation) trough plasma tafenoquine concentrations were 223 +/- 41, 127 +/- 29, 157 +/- 51. 120 +/- 24, and 88 +/- ng/ mL. Only 1 soldier developed malaria during the study. At the time of malaria diagnosis, his plasma tafenoquine concentration was 40 ng/ mL, which was similar to 3- fold lower than the trough concentrations of the other soldiers. Although low tafenoquine concentrations appear to be uncommon, additional investigations are needed to determine the relationship between plasma tafenoquine concentrations and suppression of malaria.

Assessment of volume depletion in children with malaria

Background The degree of volume depletion in severe malaria is currently unknown, although knowledge of fluid compartment volumes can guide therapy. To assist management of severely ill children, and to test the hypothesis that volume changes in fluid compartments reflect disease severity, we measured body compartment volumes in Gabonese children with malaria. Methods and Findings Total body water volume (TBW) and extracellular water volume (ECW) were estimated in children with severe or moderate malaria and in convalescence by tracer dilution with heavy water and bromide, respectively. Intracellular water volume (ICW) was derived from these parameters. Bioelectrical impedance analysis estimates of TBW and ECW were calibrated and bioelectrical impedance analysis measurements were taken daily against dilution methods, until discharge. Sixteen children had severe and 19 moderate malaria. Severe childhood malaria was associated with depletion of TBW (mean [SD] of 37 [33] ml/kg, or 6.7% [6.0%]) relative to measurement at discharge. This is defined as mild dehydration in other conditions. ECW measurements were normal on admission in children with severe malaria and did not rise in the first few days of admission. Volumes in different compartments (TBW, ECW, and ICW) were not related to hyperlactataemia or other clinical and laboratory markers of disease severity. Moderate malaria was not associated with a depletion of TBW. Conclusions Significant hypovolaemia does not exacerbate complications of severe or moderate malaria. As rapid rehydration of children with malaria may have risks, we suggest that fluid replacement regimens should aim to correct fluid losses over 12-24 h.

Detection and differentiation of Plasmodium species by polymerase chain reaction and colorimetric detection in blood samples of patients with suspected malaria

Polymerase chain reaction (PCR) is now recognized as a sensitive and specific method for detecting Plasmodium species in blood. In this Study. we tested 279 blood samples, from patients with Suspected malaria, by a PCR assay utilizing species-specific colorimetric detection. and compared the results to light microscopy. Overall, both assays were in agreement for 270 of the 279 specimens. P. vivax was detected in 131 (47.0%) specimens. P. falciparum in 64 (22.9%) specimens, P. ovale in 6 (2.1%) specimens, and P. malariae in 5 (1.8%) specimens. Both P. falciparum and P. vivax were detected in a further 10 (3.6%) specimens, and 54 (19.3%) specimens were negative by both assays. In the remaining nine specimens, microscopy either failed to detect the parasite or incorrectly identified the species present. In summary, the sensitivity, specificity and simplicity of the PCR assay makes it particularly suitable for use in a diagnostic laboratory. (C) 2004 Elsevier Inc. All rights reserved.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.

Inhibition of 19-kDa C-terminal region of merozoite surface protein-1-specific antibody responses in neonatal pups by maternally derived 19-kDa C-terminal region of merozoite surface protein-1-specific antibodies but not whole parasite-specific antibodies

Immunizing pregnant women with a malaria vaccine is one approach to protecting the mother and her offspring from malaria infection. However, specific maternal Abs generated in response to vaccination and transferred to the fetus may interfere with the infant's ability to respond to the same vaccine. Using a murine model of malaria, we examined the effect of maternal 19-kDa C-terminal region of merozoite surface protein-1 (MSP1(19)) and Plasmodium yoelii Abs on the pups' ability to respond to immunization with MSP1(19). Maternal MSPI,g-specific Abs but not A yoelii-specific Abs inhibited Ab production following MSP1(19) immunization in 2-wk-old pups. This inhibition was correlated with the amount of maternal MSP1(19) Ab present in the pup at the time of immunization and was due to fewer specific B cells. Passively acquired Ab most likely inhibited the development of an Ab response by blocking access to critical B cell epitopes. If a neonate's ability to respond to MSP1(19) vaccination depends on the level of maternal Abs present at the time of vaccination, it may be necessary to delay immunization until Abs specific for the vaccinating Ag have decreased.

Antiretrovirals as antimalarial agents

Recent studies have indicated that antiretroviral protease inhibitors may affect outcome in malarial disease. We have investigated the antimalarial activities of 6 commonly used antiretroviral agents. Our data indicate that, in addition to the previously published effects on cytoadherence and phagocytosis, the human immunodeficiency virus (HIV)-1 protease inhibitors saquinavir, ritonavir, and indinavir directly inhibit the growth of Plasmodium falciparum in vitro at clinically relevant concentrations. These findings are particularly important in light of both the high rate of malaria and HIV-1 coinfection in sub-Saharan Africa and the effort to employ highly active antiretroviral therapy in these regions.