Plasmodium Life Cycle Research Articles

Discovery of HDAC inhibitors with potent activity against multiple malaria parasite life cycle stages

In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage Plasmodium falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against Plasmodium berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages.

Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

Author SummaryMalaria, caused by Plasmodium spp. parasites, is a profound human health problem. Plasmodium parasites progress through a complex life cycle as they move between infected humans and blood-feeding mosquitoes. We know that tight regulation of calcium ion levels within the cytosol of the parasite is critical to control multiple signalling events in their life cycle. However, how these calcium levels are controlled remains a mystery. Here, we show that a single protein kinase, the cGMP-dependent protein kinase G (PKG), controls the calcium signals that are critical at three different points of the life cycle: (1) for the exit of the merozoite form of the parasite from human erythrocytes (red blood cells), (2) for the cellular activation that happens when Plasmodium sexual transmission stages are ingested by a blood-feeding mosquito, and (3) for the productive gliding of the ookinete, which is the parasite stage that invades the mosquito midgut. We provide initial evidence that the universal role of PKG relies on the production of lipid precursors which then give rise to inositol (1,4,5)-trisphosphate (IP3), a messenger molecule that serves as a signal for the release of calcium from stores within the parasite. This signalling pathway provides a potential target to block both malaria development in the human host and transmission to the mosquito vector.

Identification of Vital and Dispensable Sulfur Utilization Factors in the Plasmodium Apicoplast

Iron-sulfur [Fe-S] clusters are ubiquitous and critical cofactors in diverse biochemical processes. They are assembled by distinct [Fe-S] cluster biosynthesis pathways, typically in organelles of endosymbiotic origin. Apicomplexan parasites, including Plasmodium, the causative agent of malaria, harbor two separate [Fe-S] cluster biosynthesis pathways in the their mitochondrion and apicoplast. In this study, we systematically targeted the five nuclear-encoded sulfur utilization factors (SUF) of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria model parasite Plasmodium berghei. We show that four SUFs, namely SUFC, D, E, and S are refractory to targeted gene deletion, validating them as potential targets for antimalarial drug development. We achieved targeted deletion of SUFA, which encodes a potential [Fe-S] transfer protein, indicative of a dispensable role during asexual blood stage growth in vivo. Furthermore, no abnormalities were observed during Plasmodium life cycle progression in the insect and mammalian hosts. Fusion of a fluorescent tag to the endogenous P. berghei SUFs demonstrated that all loci were accessible to genetic modification and that all five tagged SUFs localize to the apicoplast. Together, our experimental genetics analysis identifies the key components of the SUF [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite and shows that absence of SUFC, D, E, or S is incompatible with Plasmodium blood infection in vivo.

The Plasmodium protein P113 supports efficient sporozoite to liver stage conversion in vivo

Invasive stages of Plasmodium parasites possess distinct integral and peripheral membrane proteins that mediate host cell attachment and invasion. P113 is an abundant protein in detergent-resistant high molecular weight complexes in Plasmodium schizonts, but is unusual since expression extends to gametocytes and sporozoites. In this study, we tested whether P113 performs important functions for parasite propagation in Plasmodium berghei. We show that pre-erythrocytic expression of P113 displays key signatures of upregulated in infectious sporozoites (UIS) genes, including control by the liver stage master regulator SLARP. Targeted gene deletion resulted in viable blood stage parasites that displayed no signs of blood stage growth defects. p113(−) parasites propagated normally through the life cycle until mature sporozoites, but displayed defects during natural sporozoite transmission, leading to a delay to patency in infected animals. By comparative in vitro and in vivo analysis of pre-erythrocytic development and using a xeno-diagnostic test we show that ablation of P113 results in lower sporozoite to liver stage conversion and, as a consequence, reduced merozoite output in vivo, without delaying liver stage development. We conclude that p113 is dispensable for Plasmodium life cycle progression and plays auxiliary roles during pre-erythrocytic development.

The Malarial Serine Protease SUB1 Plays an Essential Role in Parasite Liver Stage Development

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

Aminoacylation of Plasmodium falciparum tRNAAsn and Insights in the Synthesis of Asparagine Repeats

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA(Asn). However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA(Asn) accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA(Asn) in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as "tRNA sponges" and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.

Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood

Successful completion of the Plasmodium lifecycle in the mosquito vector is critical for malaria transmission. It has been documented that the fate of Plasmodium in the mosquito ultimately depends on a fine interplay of molecular mosquito factors that act as parasite agonists and antagonists. Here we investigate whether the cellular responses of the invaded midgut epithelium can also determine the parasite fate and development. We show that the parasite hood, an actin-rich structure formed around the ookinete as it exits the epithelium, is a local epithelial defence reaction observed around 60% of invading parasites. The hood co-localizes with WASP, a promoter of actin filament nucleation, suggesting that it is an active reaction of the invaded cell against invading parasites. Importantly, depletion of WASP by RNAi leads to a significant reduction in hood formation, which is consistent with the previously documented role of this gene as a potent parasite antagonist. Indeed, in mosquitoes that are either genetically selected or manipulated by RNAi to be refractory to Plasmodium, most dead parasites exhibit an actin hood. In these mosquitoes, invading ookinetes are killed by lysis or melanization while exiting the midgut epithelium. Silencing WASP in these mosquitoes inhibits the formation of the hood and allows many parasites to develop to oocysts. These data in conjunction with fine microscopic observations suggest that the presence of the hood is linked to ookinete killing through lysis.

Taking a Bite Out of Mosquitoes: A New Drug to Block Transmission of Malaria

Despite concerted control efforts and decades of research, malaria remains a global scourge and the most important parasitic disease of humans. Infections caused by the parasite Plasmodium falciparum exact a huge toll, largely in subSaharan Africa, where nearly a million children die annually from complications of malaria. Integrated campaigns such as The Malaria Control and Evaluation Program in Africa (MACEPA) have adopted comprehensive strategies including insecticide-impregnated bed nets, artemesininbased combination therapies, aggressive indoor insecticide spraying, and advanced diagnostics to reduce mortality in children by nearly 20 percent [1]. Such recent advances coupled with evolving prospects for a vaccine against malaria [2] suggest that the goal of malaria eradication may soon be attainable, using an arsenal of preventive practices. For now, chemotherapeutic measures remain the mainstay of malaria management. Currently, combination therapy with artemesinin derivatives and agents such as mefloquine, lumefantrine, or amodiaquine are first-line therapies for uncomplicated malaria in most endemic regions of the world. Such treatments can be highly effectivewithresolutionofparasitemiawhen administered soon after onset. Although artemesinin-based therapies have proven successful in many trials across Africa and SoutheastAsia, there are growing concerns about evolving resistance of P. falciparum to these agents. Recently, Phyo et al [3] reported an alarming increase in rates of artemesinin resistance amongst P. falciparum isolates frompatients seen in clinics along the Thailand-Myanmar border.Furthermore,resistanceratesinWestern Cambodia were reported to be greater than 40percent.Assays of parasite clearancewere correlated with genotypic changes in parasite populations and nearly two-thirds of the variation in parasite clearance over a 3-year period was attributable to genetic polymorphisms in populations of P. falciparum. Indeed, emergence of widespread artemesinin resistance in this region of the world underscores the pressing need to develop novel anti-malarial agents. In this issue of the Journal, Ojo et al [4] report a very exciting new prospect for drug treatment of P. falciparum. The investigators describe a new class of antimalarial compounds that target the sexual stage of Plasmodium (Figure 1), specifically the parasite’s calcium-dependent protein kinase 4 (CDPK4), a signaling molecule that is essential for exflagellation of male gametocytes. This step is a precursor to the fusion ofmale and female gametocytes, which results in zygote formation in the mosquito. The sexual phase of the Plasmodium life cycle is critical and represents a potential target for drugs that would ultimately abort transmission of parasites by mosquitoes to humans. Compound 1294, reported by Ojo et al is a synthetic inhibitor of Plasmodium CDPK4. The authors demonstrate convincingly that the synthetic CDPK4 inhibitor inhibits exflagellation of gametocytes and, by acting on a specific serine residue of the parasite, avoids cross-reactivity with mammalian kinase substrates. Furthermore, the mechanism by which this compound acts on parasites is cleverly elucidated, using transgenic lines of Plasmodium. By generating a mutant parasite with a modified target site for the enzyme, the authors demonstrate that EC50 exflagellation values are shifted relative to wild-type parasites exposed to the same inhibitor. Thus, both efficacy of compound 1294 and its mode of action are presented in this seminal article. Gametocytes of P. falciparum begin maturation in the mammalian host and are taken up during a mosquito’s blood meal (Figure 1). Fusion of the gametocytes and formation of a zygote occur in the arthropod. Therefore, a CDPK4 inhibitor, taken as a drug by a human, would disrupt the parasite life cycle within the insect and prevent formation of infectious sporozoites. For this strategy to be effective, the drug would have to be widely bioavailable and have a long half-life, because Received and accepted 15 July 2013. Correspondence: Ravi Durvasula, MD, Chief of Medicine, Raymond G. Murphy VA Medical Center, 1501 San Pedro Drive SE, Albuquerque, NM 87108 (ravi.durvasula@va.gov). The Journal of Infectious Diseases Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2013. This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: 10.1093/infdis/jit523

Design, Synthesis, and Biological and Crystallographic Evaluation of Novel Inhibitors of Plasmodium falciparum Enoyl-ACP-reductase (PfFabI)

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

Impact of climate variability on Plasmodium vivax and Plasmodium falciparum malaria in the high risk area of Yunnan Province, China

Background: Malaria remains a public health problem in the remote and poor area of Yunnan Province, China. Yunnan faces an increasing risk of imported malaria infections from Mekong-River neighboring countries. Aims: This study aimed to identify the high risk area of malaria transmission in Yunnan Province, and to estimate the effects of climatic variability on the transmission of Plasmodium vivax and Plasmodium falciparum in the identified area. Methods: We identified spatial clusters of malaria cases using spatial cluster analysis at a county level in Yunnan Province, during 2005-2010, and estimated the weekly effects of climatic factors on P.vivax and P.falciparum based on a dataset of daily malaria cases and climatic variables. A distributed lag nonlinear model (DLNM) was used to estimate the impact of temperature, relative humidity and rainfall on both types of malaria parasite up to 10-week lags. Results: The results show that the primary cluster area was identified in western Yunnan along China-Myanmar border. The relative risk (RR) in malaria caseswith a 1 °C increase in minimum temperature was statistically significant from lag 4 to lag 9 weeks with the highest RRs of 1.03 (95% CI, 1.01, 1.05) for P.vivax at a 7 week lag and1.07 (95% CI, 1.04, 1.11) for P.falciparum at a 6-week lag; the RR with a 10-mm increment in rainfall was statistically significant from lag 2 to lag 4 weeks and from lag 9 to lag 10 weeks with the highest RRs of1.03 (95% CI, 1.01, 1.05) forP.vivax at a 2-week lag and 1.04 (95% CI, 1.01, 1.06) for P.falciparum at a lag of 2 weeks; and the RR with a 10% rise in relative humidity was statistically significant from lag 3 to lag 8 weeks with the highest RRs of 1.24 (95% CI: 1.10, 1.41) for P.vivax at a 5-week lag. Conclusions: Our findings suggest that the China-Myanmar border is a high risk area for malaria transmission. Climatic factors appeared to be major determinants for malaria transmission in this area. The estimated lag effects for these factors and malaria are consistent with the life cycle of malaria parasite.

Plasmodium falciparum Hsp70-x: a heat shock protein at the host–parasite interface

Plasmodium falciparum 70 kDa heat shock proteins (PfHsp70s) are expressed at all stages of the pathogenic erythrocytic phase of the malaria parasite life cycle. There are six PfHsp70s, all of which have orthologues in other plasmodial species, except for PfHsp70-x which is unique to P. falciparum. This research highlights a number of original results obtained by a detailed bioinformatics analysis of the protein. Large-scale sequence analysis indicated the presence of an extended transit peptide sequence of PfHsp70-x which potentially directs it to the endoplasmic reticulum (ER). Further analysis showed that PfHsp70-x does not have an ER-retention sequence, suggesting that the protein transits through the ER and is secreted into the parasitophorous vacuole or beyond into the erythrocyte cytosol. These results are consistent with experimental findings. Next, possible interactions between PfHsp70-x and exported P. falciparum Hsp40s or host erythrocyte Hsp40 were interrogated by modelling and docking. Docking results indicated that interaction between PfHsp70-x and each of the Hsp40s, regardless of biological feasibility, seems equally likely. This suggests that J domain might not provide the specificity in the formation of unique Hsp70-Hsp40 complexes, but that the specificity might be provided by other domains of Hsp40s. By studying different structural conformations of PfHsp70-x, it was shown that Hsp40s can only bind when PfHsp70-x is in a certain conformation. Additionally, this work highlighted the possible dependence of the substrate-binding domain residues on the orientation of the α-helical lid for formation of the substrate-binding pocket.

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

New medicines for control and elimination of malaria

Despite being 1 of the most prevalent tropical diseases, for many years malaria was not a commercial priority for the pharmaceutical industry. However, in response to the emergence and spread of resistance to the available antimalarial drugs, there has been a renaissance in the discovery and development of new medicines to control the disease in the last few years. The persistent threat of resistance means that new molecules with novel mechanisms of action are continually required. Furthermore, the recent call for the elimination and eradication of malaria has prompted an extension of the stages of the life cycle of malaria parasites that should be targeted by new molecules.

Anopheles gambiae Circumsporozoite Protein–Binding Protein Facilitates Plasmodium Infection of Mosquito Salivary Glands

Malaria, a mosquito-borne disease caused by Plasmodium species, causes substantial morbidity and mortality throughout the world. Plasmodium sporozoites mature in oocysts formed in the mosquito gut wall and then invade the salivary glands, where they remain until transmitted to the vertebrate host during a mosquito bite. The Plasmodium circumsporozoite protein (CSP) binds to salivary glands and plays a role in the invasion of this organ by sporozoites. We identified an Anopheles salivary gland protein, named CSP-binding protein (CSPBP), that interacts with CSP. Downregulation of CSPBP in mosquito salivary glands inhibited invasion by Plasmodium organisms. In vivo bioassays showed that mosquitoes that were fed blood with CSPBP antibody displayed a 25% and 90% reduction in the parasite load in infected salivary glands 14 and 18 days after the blood meal, respectively. These results suggest that CSPBP is important for the infection of the mosquito salivary gland by Plasmodium organisms and that blocking CSPBP can interfere with the Plasmodium life cycle.

Life history focus on a malaria parasite: linked traits and variation among genetic clones

Life history theory has long been a major campaign in evolutionary ecology, but has typically focused only on animals and plants. Life history research on single-celled eukaryotic protists such as malaria parasites (Plasmodium) will offer new insights into the theory’s general utility as well as the parasite’s basic biology. For example, parasitologists have described the Plasmodium life cycle and cell types in exquisite detail, with little discussion of evolutionary issues such as developmental links between traits. We measured 10 life history traits of replicate single-genotype experimental Plasmodium mexicanum infections in its natural lizard host to identify groups of linked traits. These 10 traits formed 4 trait groups: “Rate/Peak” merges measures of growth rate and maximum parasitemia of infections; “Timing” combines time to patency and maximum parasitemia; “Growth Shape” describes the fit to an exponential growth curve; and “Sex Ratio” includes only the gametocyte sex ratio. Parasite genotype (clone) showed no effect on the life history trait groups, with the exception of gametocyte sex ratio. Therefore, variation in most life history traits among infections appears to be driven by environmental (individual host) effects. The findings support the model that life history traits are often linked by developmental constraints. Understanding why life history traits of Plasmodium are linked in this way would offer a new window into the evolution of the parasites, and also should inform public health efforts to control infection prevalence.

An Image Analysis Algorithm for Malaria Parasite Stage Classification and Viability Quantification

With more than 40% of the world’s population at risk, 200–300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays.

Loss‐of‐function analyses defines vital and redundant functions of the Plasmodium rhomboid protease family

Rhomboid-like proteases cleave membrane-anchored proteins within their transmembrane domains. In apicomplexan parasites substrates include molecules that function in parasite motility and host cell invasion. While two Plasmodium rhomboids, ROM1 and ROM4, have been examined, the roles of the remaining six rhomboids during the malaria parasite's life cycle are unknown. We present systematic gene deletion analyses of all eight Plasmodium rhomboid-like proteins as a means to discover stage-specific phenotypes and potential functions in the rodent malaria model, P. berghei. Four rhomboids (ROM4, 6, 7 and 8) are refractory to gene deletion, suggesting an essential role during asexual blood stage development. In contrast ROM1, 3, 9 and 10 were dispensable for blood stage development and exhibited no, subtle or severe defects in mosquito or liver development. Parasites lacking ROM9 and ROM10 showed no major phenotypic defects. Parasites lacking ROM1 presented a delay in blood stage patency following liver infection, but in contrast to a previous study blood stage parasites had similar growth and virulence characteristics as wild type parasites. Parasites lacking ROM3 in mosquitoes readily established oocysts but failed to produce sporozoites. ROM3 is the first apicomplexan rhomboid identified to play a vital role in sporogony.

Salivary Glands Proteins Expression ofAnopheles dirusA Fed onPlasmodium vivax-andPlasmodium falciparum-Infected Human Blood

Mosquitoes are able to adapt to feed on blood by the salivary glands which created a protein that works against the haemostasis process. This study aims to investigate the salivary glands proteins expression of 50 adult female An. dirus A mosquitoes, a main vector of malaria in Thailand, each group with an age of 5 days which were artificial membrane fed on sugar, normal blood, blood infected with P. vivax, and blood infected with P. falciparum. Then mosquito salivary gland proteins were analyzed by SDS-PAGE on days 0, 1, 2, 3, and 4 after feeding. The findings revealed that the major salivary glands proteins had molecular weights of 62, 58, 43, 36, 33, 30, and 18 kDa. One protein band of approximately 13 kDa was found in normal blood and blood infected with P. vivax fed on day 0. A stronger protein band, 65 kDa, was expressed from the salivary glands of mosquitoes fed with P. vivax- or P. falciparum-infected blood on only day 0, but none on days 1 to 4. The study shows that salivary glands proteins expression of An. dirus may affect the malaria parasite life cycle and the ability of mosquitoes to transmit malaria parasites in post-24-hour disappearance observation.

Antimalarial drug discovery: targeting the hypnozoite for new radical curative agent

Plasmodium vivax malaria remains a significant health burden in endemic areas like South East Asia, Central and South America. P. vivax infections are characterized by relapses of malaria caused by persistent liver stages of the parasite (hypnozoites) which are not present in P. falciparum infections. Currently, the only approved treatment option for the radical cure of P. vivax malaria is the 8-aminoquinoline, primaquine. However the long treatment course (two weeks) and severe side effects (hemolytic anemia in G6PD-deficeint patients) highlights the need for new chemical classes of drugs. Currently the discovery of new anti-hypnozoite drugs is limited to testing in the P. cynomolgi rhesus monkey model. However recently developed assays that allow for the testing of new chemical entities on the parasite liver-stages (schizonts) and sexual stages (gametocytes) could be leveraged to identify dual acting compounds with potental anti-hypnozoite activity. In addition, compounds which target more than one stage in the Plasmodium lifecycle would be highly valued and compliment the current arsenal of antimalarial drugs.

Antimalarial Activity of Pyrroloiminoquinones from the Australian Marine Sponge Zyzzya sp.

A new bispyrroloiminoquinone alkaloid, tsitsikammamine C (1), displayed potent in vitro antimalarial activity with IC(50) values of 13 and 18 nM against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum, respectively. Tsitsikammamine C (1) displayed selectivity indices of >200 against HEK293 cells and inhibited both ring and trophozoite stages of the malaria parasite life cycle. Previously reported compounds makaluvamines J (2), G (3), L (4), K (5) and damirones A (6) and B (7) were also isolated from the same marine sponge (Zyzzya sp.). Compounds 2-4 displayed potent growth inhibitory activity (IC(50) < 100 nM) against both P. falciparum lines and only moderate cytotoxicity against HEK293 cells (IC(50) = 1-4 μM). Makaluvamine G (3) was not toxic to mice and suppressed parasite growth in P. berghei infected mice following subcutaneous administration at 8 mg kg(-1) day(-1).