Impact of red blood cell polymorphisms on the antibody response to Plasmodium falciparum in Senegal

The evidence of protection afforded by red blood cell polymorphisms against either clinical malaria or Plasmodium falciparum blood levels varies with the study site and the type of malaria transmission. Nevertheless, no clear implication of an antibody-related effect has yet been established in the protection related to red blood cell polymorphisms. We performed a prospective study, where plasma IgG and IgG subclasses directed to recombinant proteins from the merozoite surface protein 2 (MSP2/3D7 and MSP2/FC27) and the ring-infected erythrocyte surface antigen (RESA) were determined in a cohort of 413 Senegalese children before the annual malaria transmission season. The antibody response was dependent on age, and to a lesser extent, on the village of residence. IgG3 responders to all proteins, IgG responders to RESA and MSP2/3D7, as well as IgG2 to RESA and IgG1 responders to MSP2/3D7, presented enhanced mean values of parasite density, as evaluated during an 18-month follow-up. The levels of IgG and IgG3 to MSP2/3D7 were negatively associated with the risk of occurrence of a malaria attack during the following transmission season. Compared to normal children, sickle cell trait carriers presented lower levels of IgG to MSP2/3D7. Similarly, G6PD A- girls had lower levels of IgG and IgG3 to MSP2/FC27 than did G6PD normal girls. The impact of these particular genetic polymorphisms on the modulation of the antibody response is discussed.     

Plasmodium falciparum genotype population dynamics in asymptomatic children from Senegal

In areas where malaria is endemic, infected individuals generally harbor a mixture of genetically distinct Plasmodium falciparum parasite populations. For the first time, we studied temporal variations of blood parasite densities and circulating genotypes in asymptomatic Senegalese children, at time intervals as short as 4-12 h. Twenty-one Senegalese children, presenting with an asymptomatic P. falciparum infection, were sampled eight times within three days. Parasite density was assessed by thick blood smears, and all infecting genotypes were quantified by the fragment-analysis method. Parasite densities showed dramatic fluctuations up to a 1 to 1,000 ratio, with at least one peak of parasite density. Polyclonal infections were detected in all children, with a multiplicity of infection of 5.2-6.8 genotypes per child. A single sample never reflected the full complexity of the parasite populations hosted by a given individual. Genotypes with different behaviors were detected in all children, some genotypes undergoing major fluctuations, while others were highly stable during the follow-up. A single peripheral blood sampling does not reflect the total parasite load. Repeated sampling is needed to have a more detailed scheme of parasite population dynamics and a better knowledge of the true complexity of an infection.     

High prevalence of Plasmodium malariae and Plasmodium ovale in co-infections with Plasmodium falciparum in asymptomatic malaria parasite carriers in southwestern Nigeria

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10–19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.     

Chromosome size polymorphisms in Plasmodium falciparum can involve deletions and are frequent in natural parasite populations

A comparison of independent cultured isolates of Plasmodium falciparum revealed that while chromosome number was constant, the sizes of analogous chromosomes varied widely. We show here that chromosome size polymorphisms are not generated during differentiation of the asexual blood stages, as the molecular karyotype of a cloned parasite line is constant through this part of the life cycle. Experiments using whole P. falciparum chromosomes as hybridization probes to examine polymorphisms within two independent parasite populations indicate that the polymorphisms observed here are not the consequence of large-scale interchromosomal exchanges, and imply that deletions/duplications represent one mode of generating chromosome length polymorphisms. Although the deletions probably involve repetitive DNA, we show here that structural genes for P. falciparum antigens can also be lost. Furthermore, these dramatic size polymorphisms occur not only in cultured lines of P. falciparum, but with surprising frequency in natural malarial infections.     

Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum

Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long-term survival of the parasite.     

Plasmodium falciparum: mapping genes to nine parasite chromosomes

Using pulsed field gel electrophoresis with pulse time of 120 sec, eight chromosomal DNA molecules from clone 7G8 of the Plasmodium falciparum Brazilian isolate IMTM22 were resolved. A ninth chromosomal molecule which did not enter the gel was identified at the slot by hybridization to two DNA probes and by restriction enzyme analysis. Thirteen parasite DNA sequences were mapped to the nine chromosomes, with at least one sequence mapped to each chromosome. The restriction enzyme NotI appeared to produce only one cut in the entire IMTM22 genome.     

Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite

Selected aspects of the metabolism of Plasmodium falciparum are reviewed, but conclusions based on the study of other species of plasmodia are intentionally not included since these may not be applicable. The parasites increase glucose consumption 50-100 fold as compared to uninfected red cells; most of the glucose is metabolized to lactic acid. The parasite contains a complete set of glycolytic enzymes. Some enzymes such a hexokinase, enolase and pyruvate kinase are vastly increased over corresponding levels in uninfected red cells. However, the pathway for synthesizing 2,3-diphosphoglycerate (2,3-DPG) is absent. Parasitized red cells show a decline in the concentration of 2,3-DPG which may function as an inhibitor for certain essential enzyme pathways. Pentose shunt activity is increased in absolute terms, but as a percent of total glucose consumption, there is a decrease during parasite infection of the red cell. The parasite contains a gene for G6PD and can produce a small quantity of parasite-encoded enzyme. It is not clear if the production of this enzyme can be up-regulated in G6PG deficient host red cells. The NADPH normally produced by the pentose shunt can be obtained from other parasite pathways (such as glutamate dehydrogenase). NADPH may subserve additional needs in the infected red cell such as driving diribonucleotide reductase activity--a rate limiting enzyme in DNA synthesis. The role of NADPH in protecting the parasite-red cell system against oxidative stress (via glutathione reduction) remains controversial. Parasitized red cells contain about 10 times more NAD(H) than uninfected red cells, but the NADP(H) content is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutathione--functions and metabolism in the malarial parasite Plasmodium falciparum

When present as a trophozoite in human erythrocytes, the malarial parasite Plasmodium falciparum exhibits an intense glutathione metabolism. Glutathione plays a role not only in antioxidative defense and in maintaining the reducing environment of the cytosol. Many of the known glutathione-dependent processes are directly related to the specific lifestyle of the parasite. Reduced glutathione (GSH) supports rapid cell growth by providing electrons for deoxyribonucleotide synthesis and it takes part in detoxifying heme, a product of hemoglobin digestion. Free radicals generated in the parasite can be scavenged in reaction sequences involving the thiyl radical GS* as well as the thiolate GS-. As a substrate of glutathione S-transferase, glutathione is conjugated to non-degradable compounds including antimalarial drugs. Furthermore, it is the coenzyme of the glyoxalase system which detoxifies methylglyoxal, a byproduct of the intense glycolysis taking place in the trophozoite. Proteins involved in GSH-dependent processes include glutathione reductase, glutaredoxins, glyoxalase I and II, glutathione S-transferases, and thioredoxins. These proteins, as well as the ATP-dependent enzymes of glutathione synthesis, are studied as factors in the pathophysiology of malaria but also as potential drug targets. Methylene blue, an inhibitor of the structurally known P. falciparum glutathione reductase, appears to be a promising antimalarial medication when given in combination with chloroquine.     

Centrins, cell cycle regulation proteins in human malaria parasite Plasmodium falciparum

Molecules and cellular mechanisms that regulate the process of cell division in malaria parasites remain poorly understood. In this study we isolate and characterize the four Plasmodium falciparum centrins (PfCENs) and, by growth complementation studies, provide evidence for their involvement in cell division. Centrins are cytoskeleton proteins with key roles in cell division, including centrosome duplication, and possess four Ca(2+)-binding EF hand domains. By means of phylogenetic analysis, we were able to decipher the evolutionary history of centrins in eukaryotes with particular emphasis on the situation in apicomplexans and other alveolates. Plasmodium possesses orthologs of four distinct centrin paralogs traceable to the ancestral alveolate, including two that are unique to alveolates. By real time PCR and/or immunofluorescence, we determined the expression of PfCEN mRNA or protein in sporozoites, asexual blood forms, gametocytes, and in the oocysts developing inside mosquito mid-gut. Immunoelectron microscopy studies showed that centrin is expressed in close proximity with the nucleus of sporozoites and asexual schizonts. Furthermore, confocal and widefield microscopy using the double staining with alpha-tubulin and centrin antibodies strongly suggested that centrin is associated with the parasite centrosome. Following the episomal expression of the four PfCENs in a centrin knock-out Leishmania donovani parasite line that exhibited a severe growth defect, one of the PfCENs was able to partially restore Leishmania growth rate and overcome the defect in cytokinesis in such mutant cell line. To our knowledge, this study is the first characterization of a Plasmodium molecule that is involved in the process of cell division. These results provide the opportunity to further explore the role of centrins in cell division in malaria parasites and suggest novel targets to construct genetically modified, live attenuated malaria vaccines.     

Mitosis in the human malaria parasite Plasmodium falciparum

Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes.     

Genetic mapping in the human malaria parasite Plasmodium falciparum

The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.     

Helicases involved in splicing from malaria parasite Plasmodium falciparum

An interesting element of eukaryotic genomes is the large quantity of non-coding intervening sequences commonly known as introns, which regularly interrupt functional genes and therefore must be removed prior to the use of genetic information by the cell. After splicing, the mature RNA is exported from the nucleus to the cytoplasm. Any error in the process of recognition and removal of introns, or splicing, would lead to change in genetic message and thus has potentially catastrophic consequences. Thus splicing is a highly complex essential step in eukaryotic gene expression. It takes place in spliceosome, which is a dynamic RNA-protein complex made of snRNPs and non-snRNP proteins. The splicing process consists of following sequential steps: spliceosome formation, the first transesterification and second transesterification reactions, release of the mature mRNA and recycling of the snRNPs. The spliceosomal components produce a complex network of RNA-RNA, RNA-protein and protein-protein interactions and spliceosome experience remodeling during each splicing cycle. Helicases are essentially required at almost each step for resolution of RNA-RNA and/or RNA-protein interactions. RNA helicases share a highly conserved helicase domain which includes the motif DExD/H in the single letter amino acid code. This article will focus on members of the DExD/H-box proteins involved specially in splicing in the malaria parasite Plasmodium falciparum.     

Plasmodium falciparum clinical malaria: lessons from longitudinal studies in Senegal

Development of new antimalaria strategies and particularly vaccines, needs an in-depth understanding of the relationships between transmission, infection, immunity, morbidity and mortality. The intensive and longitudinal collection of entomological, parasitological and clinical data from the Senegalese populations of Dielmo (250-300 inhabitants), exposed to a perennial and intense transmission (about 200 infective bites/person/year) and of Ndiop (300-350 inhabitants) exposed to a seasonal transmission (about 20 infective bites/person/year), allows to respond to many questions about this subject. The acquisition of an antimalaria immunity as one gets older appears to reduce parasite density, complexity of infection, risk of new patent infection after a suppressive treatment but does not reduce the prevalence (as assessed by PCR) of infection which is commonly chronic and asymptomatic. The existence of a pyrogenic threshold effect of parasitaemia allows the individual diagnosis of malaria attacks. P. falciparum genotyping suggests that successive malaria attacks are due to distinct recently inoculated parasite populations that multiply initially without restriction, a dominant population is generally responsible of the clinical manifestations and all new populations do not trigger systematically attacks. The initial intensity of clinical manifestations does not differ perceptibly among children and adults, is not related to the duration of the attacks, does not allow the distinction between several types of attacks, is not predictive of their severity, and the clearance of parasites and manifestations is longer among youngest persons. The risk of malaria attacks is lower as one gets older and among carriers of AS haemoglobin, is higher when transmission increases and during pregnancy up to three months after delivery, and vary between children. The risk of malaria attack per infective bite is negatively related to the intensity of transmission. Because of their high sensitivity in malaria case detection, this type of small community-based studies are powerful and useful for the identification of protective immunological mechanisms as well as for testing rapidly and cheaply the clinical efficacy of any intervention such as antimalarial vaccines and drug therapy or prophylaxis. As a lot of vaccine candidates and drug combinations will be screened or tested in the perspective of the 'Roll-Back Malaria' programme, more attention must be given to longitudinal studies of this type.     

Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum

Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high‐affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes.     

Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum

We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy.     

A Plasmodium falciparum host-targeting motif functions in export during blood stage infection of the rodent malarial parasite Plasmodium berghei

Plasmodium falciparum (P. falciparum) secretes hundreds of proteins--including major virulence proteins--into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte.     

Adenylyl and guanylyl cyclases from the malaria parasite Plasmodium falciparum

Completion of several malaria parasite genome sequences and advances in Plasmodium gene manipulation technology, will lead to significant advances in our knowledge of the biology of these organisms. Biochemical analysis of the cyclic nucleotide signalling pathways of P. falciparum has provided important information on malaria parasite development. The Plasmodium purine nucleotide cyclase enzymes have extremely unusual structures and the regulatory mechanisms controlling parasite enzyme activity are distinct from those operating on the analogous host molecules. Study of these enzymes could therefore lead to novel strategies for anti-malarial intervention in addition to providing unique insights into the intriguing biology of the parasite.