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Small-Saunders et al. uncovered a new facet of artemisinin resistance in Plasmodium in which parasites use a previously underexplored arm of stress response mechanisms. Through altered epitranscriptomic modifications on tRNA, changed translation patterns adapt resistant cells to facilitate entry into a quiescent-like state which provides the parasite an escape from many drugs. [ABSTRACT FROM AUTHOR]

In 2004 the first annual BioMalPar meeting was held at EMBL Heidelberg, bringing together researchers from around the world with the goal of building connections between malaria research groups in Europe. Twenty years on it is time to reflect on what was achieved and to look ahead to the future. [ABSTRACT FROM AUTHOR]

Female anopheline mosquito reproduction is intimately linked to the Plasmodium sporogonic cycle, whereby malaria parasites ostensibly compete for the same resources required for mosquito egg development. However, in a recent study, Werling and colleagues (Cell 2019;177:315–325) uncovered a parasitic strategy supporting coexistence, exploiting mosquito nutrients without affecting mosquito fitness and reproductivity. [ABSTRACT FROM AUTHOR]

The article discusses the mechanisms employed by the blood stream plasmodium for the regulation of antigenic variation and to commit to gametocytogenesis. Topics discussed include the view of the gene expression at the intraerythrocytic developmental cycle (IDC), the initiation of chromatin regulators upon the transformation of sporozoite in the hepatocyte, and the bet hedging process.

Homeostasis of the trace element copper is essential to all eukaryotic life. Copper serves as a cofactor in metalloenzymes and catalyses electron transfer reactions as well as the generation of potentially toxic reactive oxygen species. Here, we describe the functional characterization of an evolutionarily highly conserved, predicted copper-transporting P-type ATPase ( CuTP) in the murine malaria model parasite Plasmodium berghei. Live imaging of a parasite line expressing a fluorescently tagged CuTP demonstrated that CuTP is predominantly located in vesicular bodies of the parasite. A P. berghei loss-of-function mutant line was readily obtained and showed no apparent defect in in vivo blood stage growth. Parasite transmission through the mosquito vector was severely affected, but not entirely abolished. We show that male and female gametocytes are abundant in cutp− parasites, but activation of male microgametes and exflagellation were strongly impaired. This specific defect could be mimicked by addition of the copper chelator neocuproine to wild-type gametocytes. A cross-fertilization assay demonstrated that female fertility was also severely abrogated. In conclusion, we provide experimental genetic and pharmacological evidence that a healthy copper homeostasis is critical to malaria parasite fertility of both genders of gametocyte and, hence, to transmission to the mosquito vector. [ABSTRACT FROM AUTHOR]

Abstract: The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp − parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. [Copyright &y& Elsevier]

Abstract: The SIR2 family of NAD+-dependent protein deacetylases, collectively called sirtuins, has been of central interest due to their proposed roles in life-span regulation and ageing. Sirtuins are one group of environment sensors of a cell interpreting external information and orchestrating internal responses at the sub-cellular level, through participation in gene regulation mechanisms. Remarkably conserved across all kingdoms of life SIR2 proteins in several protozoan parasites appear to have both conserved and intriguing unique functions. This review summarises our current knowledge of the members of the sirtuin families in Apicomplexa, including Plasmodium, and other protozoan parasites such as Trypanosoma and Leishmania. The wide diversity of processes regulated by SIR2 proteins makes them targets worthy of exploitation in anti-parasitic therapies. [Copyright &y& Elsevier]

The Molecular Approaches to Malaria 2004 meeting provided an opportunity to see the impressive progress in all research fields and in the four years since the previous Molecular Approaches to Malaria meeting, when much of the Plasmodium falciparum genome sequence was already available. Study of the part of the Plasmodium life cycle associated with transmission through the vector, which begins with the commitment of blood-stage forms to sexual development, has been especially fruitful. This success is a result of several reasons including: (i) the availability of the genome sequence; (ii) the availability of good animal models that allow parasite culture and facile in vivo studies of many of the life cycle stages involved in transmission; (iii) the availability of genetic manipulation technologies for the animal models of malaria, as well as P. falciparum; and (iv) the ability to study lethal gene knockouts at this stage of the life cycle. [Copyright &y& Elsevier]

Malaria parasites must invade the erythrocytes of its host, to be able to grow and multiply. Having depleted the host cell of its nutrients, the parasites break out to invade new erythrocytes. In this issue of Cell, discover a new organelle, the exoneme, that contains a protease SUB1, which helps the parasite to escape from old erythrocytes and invade new ones. [Copyright &y& Elsevier]

The life cycle of the malaria parasite (Plasmodium) is remarkably complex. Malaria parasites must engage in highly specific and varied interactions with cell types of both the mammalian host and the mosquito vector. In this issue of Cell, Tolia et al. (2005) report detailed molecular insights into an intimate interaction between a malaria parasite protein and its host cell receptor that enables the parasite to invade erythrocytes. [Copyright &y& Elsevier]

The European Virtual Institute for Malaria Research (EVIMalaR) is at risk of losing funding, which could be a major set-back for the European malaria research community. [ABSTRACT FROM AUTHOR]

The authors comments on a report within the issue by A. Molina-Cruz and team on the discovery of the glycosylphosphatidylinositol-anchored membrane protein PFS47 gene in the Plasmodium falciparum malaria protozoa, which allows the parasite to avoid the innate immune response (IIR) of its mosquito vectors to enable transmission to humans. The researchers suggest that PFS47 prevents recognition by the mosquito complement pathway through inhibition of free radical nitration and lysis. Also discussed is the potential role of PFS47 in complement binding to Plasmodium ookinetes.

The article presents information regarding gene expression microarray profiles of Plasmodium (P.) falciparum during the different stages of its complex life cycle. The three principle stages of P. falciparum development take place in the liver cells and red blood cells of the human host and the tissues of the mosquito vector. Researchers examined the gene expression profiles of P. falciparum at nine different developmental time points. In contrast, other researchers concentrated their efforts on the erythrocytic stages of development only, examining the gene expression profiles of P. falciparum every hour for the duration of the 48-hour blood stage. The emphasis of both groups on bloodstage parasites is desirable because these forms cause malaria-associated pathology and are the target of the majority of vaccine and drug development programs.

Examines the clinical implications of research on genomics and malaria control. Incidence of the disease around the world; New opportunities for prevention and eradication with recent discoveries on the genomic sequences of the most important malaria parasite, the mosquito vector, and the human being; Belief that the world need not wait for new technology but should make better use of the old; Challenge which is to provide affordable, deliverable strategies and products that will protect and cure the poorest people in developing countries; Suggestions of vector-targeted strategies; Suggestions for strategies that target human infection; Necessity of a public framework to involve affected communities in formulating new control strategies.

Responds to the readers comments on the article published in a 1999 issue of 'Nature Medicine' about malaria vaccine research. Interviews of members of the malaria research community during the research; Lack of support from the European Commission on the vaccine research.

Comments on the news article published in a 1999 issue of 'Nature Medicine,' magazine about malaria vaccine research. Impression of a competition between European and American malaria researchers and a discontinuation of clinical testing in Europe; Objection to the article's political connotation.

Malaria continues to impose a significant disease burden on low- and middle-income countries in the tropics. However, revolutionary progress over the last 3 years in nucleic acid sequencing, reverse genetics, and post-genome analyses has generated step changes in our understanding of malaria parasite (Plasmodium spp.) biology and its interactions with its host and vector. Driven by the availability of vast amounts of genome sequence data from Plasmodium species strains, relevant human populations of different ethnicities, and mosquito vectors, researchers can consider any biological component of the malarial process in isolation or in the interactive setting that is infection. In particular, considerable progress has been made in the area of population genomics, with Plasmodium falciparum serving as a highly relevant model. Such studies have demonstrated that genome evolution under strong selective pressure can be detected. These data, combined with reverse genetics, have enabled the identification of the region of the P. falciparum genome that is under selective pressure and the confirmation of the functionality of the mutations in the kelch13 gene that accompany resistance to the major frontline antimalarial, artemisinin. Furthermore, the central role of epigenetic regulation of gene expression and antigenic variation and developmental fate in P. falciparum is becoming ever clearer. This review summarizes recent exciting discoveries that genome technologies have enabled in malaria research and highlights some of their applications to healthcare. The knowledge gained will help to develop surveillance approaches for the emergence or spread of drug resistance and to identify new targets for the development of antimalarial drugs and perhaps vaccines. [ABSTRACT FROM AUTHOR]

Since the publication of the sequence of the genome of Plasmodium falciparum, the major causative agent of human malaria, many post-genomic studies have been completed. Invaluably, these data can now be analysed comparatively owing to the availability of a significant amount of genome-sequence data from several closely related model species of Plasmodium and accompanying global proteome and transcriptome studies. This review summarizes our current knowledge and how this has already been — and will continue to be — exploited in the search for vaccines and drugs against this most significant infectious disease of the tropics. [ABSTRACT FROM AUTHOR]

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, Plasmodium berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in Toxoplasma gondii if not its role in IMC formation and implicate it in microneme function. Author summary: According to the WHO there was estimated to be over 200 million cases of malaria in 2017 and nearly half a million deaths. The disease is caused by specific species of Plasmodium which are passed between human hosts by a mosquito vector. In order to transmit through the mosquito the single-celled parasite undergoes many developmental changes as it morphs from non-motile blood forms to become a polarised and motile ookinete in the mosquito midgut. Transport of proteins within the cell during these critical morphological transitions relies on specific endosome vesicles to correctly target proteins within the parasite. We investigated the role of the RAB11A protein which is known to be involved in endosomal vesicle targeting to generate cellular polarity in other organisms. Because RAB11A is also essential for parasite growth in the mammalian host we used a promoter swap system to specifically switch off RAB11A in the sexual transmission stages. In the absence of RAB11A parasites were unable to form elongated, motile ookinetes and were unable to pass through the mosquito. Interestingly the parasites were able to form some of the (polarising) structures specific to ookinetes however full morphological transformation did not occur and the parasites were not motile. We show that although proteins are still delivered to the parasite surface, secretion is impaired and that the mutant parasites are smaller despite obvious microtubule formation implying that there is a deficit in delivery of membrane to the surface. [ABSTRACT FROM AUTHOR]

The article presents a study on the behavior of Plasmodium falciparum gametocytes in the hematopoietic environment of the bone marrow. The formation of preferential homing of early gametocyte across the intact vascular barrier of the bone marrow and the spleen early during infection is demonstrated in intravital microscopy. It found that high deformability is shown by mature gametocytes while entering and exiting the intact vascular barrier.

Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite's own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles. [Display omitted] • ATZ links ART to an anti-malarial PI • Malaria-activated ATZ installs the PI on parasite proteins • ATZ overcomes resistance to both its components • ATZ suppresses recrudescence of ART-resistant parasites in mice Zhan et al. combined two anti-malarial agents—an artemisinin and a proteasome inhibitor—into a single compound called ATZ, which has an additional anti-malarial mechanism of action beyond that of its components. ATZ killed malaria resistant to either component or both. [ABSTRACT FROM AUTHOR]

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design. [ABSTRACT FROM AUTHOR]

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites. [ABSTRACT FROM AUTHOR]

Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage. [ABSTRACT FROM AUTHOR]

Background Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function. Results We have greatly improved the genome assemblies of PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilized it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the 'Plasmodium interspersed repeat genes' (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family. Conclusions Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation. [ABSTRACT FROM AUTHOR]

Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert− mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to identify telomerase inhibitors to induce parasite cell death. [ABSTRACT FROM AUTHOR]

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system. [ABSTRACT FROM AUTHOR]

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl- ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores. [ABSTRACT FROM AUTHOR]

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines. [ABSTRACT FROM AUTHOR]

Summary Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a 'promoter-swap' approach whereby the fact-l promoter was replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. 'Promoter swapping' is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development. [ABSTRACT FROM AUTHOR]

Abstract: Plasmodium falciparum malaria is the most important parasitic disease worldwide, responsible for an estimated 1 million deaths annually. Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. PGMases are responsible for the interconversion between 2 and 3-phosphoglycerate, an intermediate step in the glycolysis pathway. We have determined the crystal structures of one of the P. falciparum''s PGMases (PfPGM2) and a functionally distinct phosphoglycerate mutase from Cryptosporidium parvum, a related apicomplexan parasite. We performed sequence and structural comparisons between the two structures, another P. falciparum enzyme (PfPGM1) and several other PGM family members from other organisms. The comparisons revealed a distinct conformation of the catalytically active residues not seen in previously determined phosphoglycerate mutase structures. Furthermore, characterization of their enzymatic activities revealed contrasting behaviors between the PfPGM2 and the classical cofactor-dependent PGMase from C. parvum, clearly establishing PfPGM2 as a phosphatase with a residual level of mutase activity. Further support for this function attribution was provided by our structural comparison with previously characterized PGM family members. Genetic characterization of PGM2 in the rodent parasite Plasmodium berghei indicated that the protein might be essential to blood stage asexual growth, and a GFP tagged allele is expressed in both blood and zygote ookinete development and located in the cytoplasm. The P. falciparum PGM2 is either an enzyme implicated in the phosphate metabolism of the parasite or a regulator of its life cycle. [Copyright &y& Elsevier]

Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317+ dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317+ dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development. [ABSTRACT FROM AUTHOR]

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy. [ABSTRACT FROM AUTHOR]

Background: The genome of a number of species of malaria parasites (Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo. Results: We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated. Conclusion: These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δpcrmp3 and Δpcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites. [ABSTRACT FROM AUTHOR]

The RMgm database, www.pberghei.eu, is a web-based, manually curated, repository containing information on genetically modified rodent-malaria parasites. It provides easy and rapid access to information on the genotype and phenotype of mutant and reporter parasites. The database also contains information on unpublished mutants without a clear phenotype and negative trials to disrupt genes. Information can be searched using pre-defined key features, such as phenotype, life-cycle stage, gene model, gene-tags and mutations. The information relating to the mutants is reciprocally linked to PlasmoDB and GeneDB. Access to mutant-parasite information, and gene function/ontology inferred from mutant phenotypes provides a timely resource aimed at enhancing research into Plasmodium gene function and (systems) biology. [ABSTRACT FROM AUTHOR]

Background: The genome of a number of species of malaria parasites (Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo. Results: We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated. Conclusion: These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-γ in malaria infection. [ABSTRACT FROM AUTHOR]

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates. [ABSTRACT FROM AUTHOR]

A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocytemutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria. [ABSTRACT FROM AUTHOR]

The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 39, 59-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase δ (PDEδ), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase β (GCβ), showing that in ookinetes GCβ is an important source for cGMP, and that PDEδ is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito. [ABSTRACT FROM AUTHOR]

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes – the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue , is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Δ mdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites. [ABSTRACT FROM AUTHOR]

Abstract: Invasive forms of apicomplexan parasites contain secretory organelles (which may include micronemes, rhoptries or dense granules), the contents of which mediate invasion of host cells. Only few rhoptry proteins have been identified in Plasmodium and then only in merozoites and none in the sporozoite or ookinete. Epitope-tagged proteins (with either green fluorescent protein or C-MYC) were used to analyse the expression and cellular localisation of a known Plasmodium rhoptry protein (RAP2/3) and putative homologues of two Toxoplasma rhoptry proteins (rhoptry neck protein 2, RON2 and putative rhoptry protein 2, PRP2) at different stages across the life cycle. This analysis showed correct targeting to the merozoite rhoptries of GFP-tagged RAP2/3 and, for the first time, a distinct apical fluorescence pattern in sporozoites indicating a rhoptry location. In addition, tagged PBRON2 and PBPRP2 also show a merozoite rhoptry localisation similar to that of RAP2/3. RON2 is expressed in sporozoites and has the same timing of expression and location as RAP2/3. While PRP2 is also expressed in sporozoites, both its pattern of expression and location differ from RON2 and RAP2/3. None of the tagged proteins were detected in ookinetes, which is in agreement with the proposed lack of rhoptries in this third invasive form of Plasmodium. The analysis of tagged rhoptry proteins reveals new insights into the role of these proteins in host-cell invasion in different malarial ‘zoites’ and will facilitate more detailed studies into the role of rhoptries in establishing an infection of not only red blood cell but also the hepatocytes. [Copyright &y& Elsevier]

Gene expression in Plasmodium parasites undergoes significant changes in each developmental stage, but the transcription factors (TFs) regulating these changes have not been identified. We report here a Plasmodium TF (AP2-O) that activates gene expression in ookinetes, the mosquito-invasive form, and has a DNA-binding domain structurally related to that of a plant TF, Apetala2 (AP2). AP2-O mRNA is pre-synthesized by intraerythrocytic female gametocytes and translated later during ookinete development in the mosquito. The Plasmodium TF activates a set of genes, including all genes reported to be required for midgut invasion, by binding to specific six-base sequences on the proximal promoter. These results indicate that AP2 family TFs have important roles in stage-specific gene regulation in Plasmodium parasites. [ABSTRACT FROM AUTHOR]

Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs2 parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs2 parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito. [ABSTRACT FROM AUTHOR]

Abstract: Plasmodium encodes a family of six secreted multi-domain adhesive proteins, termed PCCps, which are released from gametocytes during emergence within the mosquito midgut. The expression and cellular localization of PCCp proteins predict a role either in gametocyte development or within the mosquito midgut during the transition from gametes into the ookinete stage. However, mutant parasites lacking expression of any single PCCp protein show a phenotype at the oocyst stage with a failure of oocyst maturation and sporozoite formation. In this study we investigated the stage-specific transcription of the PCCp genes of the rodent malaria parasite, Plasmodium berghei, and analyzed their promoter activities. Transcript expression analysis by quantitative real time RT-PCR showed that as in the human malaria parasite, Plasmodium falciparum, all PbCCp genes are predominantly transcribed in the gametocyte stage with a low level of transcription in the oocyst stage. Transgenic P. berghei parasites that contain the reporter protein GFP driven by the promoter regions of PbCCps showed pronounced GFP expression exclusively in gametocytes, in agreement with the RT-PCR data. To determine whether functional redundancies of different PCCp family members could explain the lack of a phenotype in gametocytes or gametes in single knockout mutant parasites, double gene null mutant P. berghei parasites were generated lacking either PCCp1 and PCCp3, or PCCp1 and PCCp4. The phenotype of these double knockout mutants was similar to that observed for single gene knockout mutants and manifest at the oocyst rather than the gametocyte or other stages within the mosquito midgut lumen. [Copyright &y& Elsevier]