Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

The development of an electroporation based transfection method for Plasmodium falciparum has been very successful for the study of some genes but its efficiency remains very low. While alternative approaches have been documented, electroporation of infected red blood cells generally remains the method of choice for introducing DNA into P. falciparum. In this paper we compare four published transfection techniques in their ability to achieve stable transfections.     

Construction of high-complexity combinatorial phage display peptide libraries

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.     

Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme

Plasmodium falciparum, the causative agent of human malaria, is totally dependent on de novo pyrimidine biosynthetic pathway. A gene encoding P. falciparum dihydroorotase (pfDHOase) was cloned and expressed in Escherichia coli as monofunctional enzyme. PfDHOase revealed a molecular mass of 42 kDa. In gel filtration chromatography, the major enzyme activity eluted at 40 kDa, indicating that it functions in a monomeric form. This was similarly observed using the native enzyme purified from P. falciparum. Interestingly, kinetic parameters of the enzyme and inhibitory effect by orotate and its 5-substituted derivatives parallel that found in mammalian type I DHOase. Thus, the malarial enzyme shares characteristics of both type I and type II DHOases. This study provides the monofunctional property of the parasite DHOase lending further insights into its differences from the human enzyme which forms part of a multifunctional protein.     

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)     

Plasmodium-mosquito interactions,phage display libraries and transgenic mosquitoes impaired for malaria transmission

Malaria continues to kill millions of people every year and new strategies to combat this disease are urgently needed. Recent advances in the study of the mosquito vector and its interactions with the malaria parasite suggest that it may be possible to genetically manipulate the mosquito in order to reduce its vectorial capacity. Here we review the advances made to date in four areas: (1) the introduction of foreign genes into the mosquito germ line; (2) the characterization of tissue-specific promoters; (3) the identification of gene products that block development of the parasite in the mosquito; and (4) the generation of transgenic mosquitoes impaired for malaria transmission. While initial results show great promise, the problem of how to spread the blocking genes through wild mosquito populations remains to be solved.     

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.     

Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.     

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.     

Toward quantitative genetic analysis of host and parasite traits in the manifestations of Plasmodium falciparum malaria

The complex human and parasite determinants that influence disease severity in Plasmodium falciparum malaria reflect thousands of years of selective pressure. Emerging genetic and genomic resources offer the prospect of unraveling interactions of these determinants.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

Proteomics of the human malaria parasite Plasmodium falciparum

The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.     

A Plasmodium falciparum lysophospholipase regulates host fatty acid flux via parasite lipid storage to enable controlled asexual schizogony

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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.