Curation of the Plasmodium falciparum genome

The malaria genome has proved invaluable to researchers worldwide in the continuing fight against malaria by stimulating and underpinning molecular approaches in gene expression studies, vaccine and drug discovery research, and by providing data to facilitate hypothesis-driven research. The combination of in silico and experimental investigations has already yielded dividends by strengthening our understanding of the many facets of the malaria parasite Plasmodium falciparum. The recently initiated curation of the genome resource is a vital investment for maintaining and enhancing the use of this genomic information in the post-genomic era.     

Antimalarial drug targets in Plasmodium falciparum predicted by stage-specific metabolic network analysis

Background  

Despite enormous efforts to combat malaria the disease still afflicts up to half a billion people each year of which more than one million die. Currently no approved vaccine is available and resistances to antimalarials are widely spread. Hence, new antimalarial drugs are urgently needed.     

The putative mitochondrial genome of Plasmodium falciparum

Intraerythrocytic stages of mammalian malarial parasites employ glycolysis for energy production but some aspects of mitochondrial function appear crucial to their survival since inhibitors of mitochondrial protein synthesis and electron transport have antimalarial effects. Investigations of the putative mitochondrial genome of Plasmodium falciparum have detected organellar rRNAs and tRNAs encoded by a 35 kb circular DNA. Some features of the organization and sequence of the rRNA genes are reminiscent of chloroplast DNAs. The 35 kb DNA also encodes open reading frames for proteins normally found in chloroplast but not mitochondrial genomes. An apparently unrelated 6 kb tandemly repeated element which encodes two mitochondrial protein coding genes and fragments of rRNA genes is also found in malarial parasites. The malarial mitochondrial genome thus appears quite unusual. Further investigations are expected to provide insights into the possible functional relationships between these molecules and perhaps their evolutionary history.     

Detection of homologous oncogene sequences in the genome of Plasmodium falciparum

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozoan Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.     

Tracing the dawn of Plasmodium falciparum with mitochondrial genome sequences

Until recently, little light had been shed on the murky origins of human malaria. Did Plasmodium falciparum, the most virulent malaria parasite, emerge as a common pathogen only in the past few thousand years, as suggested by some analyses of its nucleotide sequence diversity? Or, was it an ancient scourge of early humans >100 000 years ago, as suggested by others? A recent study, using complete mitochondrial DNA sequence polymorphism data and new analytical methods, points to an intermediate date of origin and expansion out of Africa. Subsequent population growth in each continent is less well resolved.     

Host chaperones are recruited in membrane-bound complexes by Plasmodium falciparum.

The ability of malarial parasite to deploy proteins at the surface of infected erythrocytes is well known. After their synthesis within the parasite, the cargo proteins are exported from the parasite and carried across the erythrocyte cytoplasm to be delivered at the erythrocyte surface. Our knowledge about the mechanisms involved in this complex trafficking path is limited. We have addressed the involvement of chaperones in traffic across erythrocyte cytoplasm. Our analyses of the chaperones available to the parasite indicated that none of the reported chaperones of the parasite origin are present in the erythrocyte cytoplasm. The chaperones of the host (Hsp70, Hsp90, Hop60), on the other hand, were readily detected in the erythrocyte cytosol. Hypotonic lysis and detergent solubilization experiments indicated that unlike their soluble nature in normal erythrocytes, host chaperones are recruited in membrane-bound, detergent-resistant complexes in infected cells. The association of host-Hsp70 with detergent-resistant complexes was ATP-dependent. Importantly, host chaperones could be detected in knob-enriched fractions and could be cross-linked to the knob subunit, PfHRP1, in a large complex at the surface of the infected erythrocytes. Our results implicate host chaperones in the assembly of parasite proteins such as knob subunits at the erythrocyte surface.     

Chimeric rRNAs containing the GTPase centers of the developmentally regulated ribosomal rRNAs of Plasmodium falciparum are functionally distinct.

The human malaria parasite, Plasmodium falciparum, maintains at least two distinct types, A and S, of developmentally controlled ribosomal RNAs. To investigate specific functions associated with these rRNAs, we replaced the Saccharomyces cerevisiae GTPase domain of the 25S rRNA with GTPase domains corresponding to the Plasmodium A- and S-type 28S rRNAs. The A-type rRNA differs in a single nonconserved base pair from the yeast GTPase domain. The S-type rRNA GTPase domain has three additional changes in highly conserved residues, making it unique among all known rRNA sequences. The expression of either A- or S-type chimeric rRNA in yeast increased translational accuracy. Yeast containing only A-type chimeric rRNA and no wild-type yeast rRNA grew at the wild-type level. In contrast, S-type chimeric rRNA severely inhibited growth in the presence of wild-type yeast rRNA, and caused lethality in the absence of the wild-type yeast rRNA. We show what before could only be hypothesized, that the changes in the GTPase center of ribosomes present during different developmental stages of Plasmodium species can result in fundamental changes in the biology of the organism.     

The physics of DNA and the annotation of the Plasmodium falciparum genome

A gene identification procedure is formulated, based on large-scale structural analyses of genomic sequences. The structural property is the physical - thermal - stability of the DNA double-helix, as described by the classical helix-coil model. The analyses are detailed for the Plasmodium falciparum genome, which represents one of the most difficult cases for the gene identification problem (notably because of the extreme AT-richness of the genome). In this genome, the coding domains (either uninterrupted genes or exons in split genes) are accurately identified as regions of high thermal stability. The conclusion is based on the study of the available cloned genes, of which 17 examples are described in detail. These examples demonstrate that the physical criterion is valid for the detection of coding regions whose lengths extend from a few base pairs up to several thousand base pairs. Accordingly, the structural analyses can provide a powerful and convenient tool for the identification of complex genes in the P. falciparum genome. The limits of such a scheme are discussed. The gene identification procedure is applied to the completely sequenced chromosomes (2 and 3), and the results are compared with the database annotations. The structural analyses suggest more or less extensive revision to the annotations, and also allow new putative genes to be identified in the chromosome sequences. Several examples of such new genes are described in detail.     

Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.     

Detergent-resistant erythrocyte membrane rafts are modified by a Plasmodium falciparum infection

Detergent resistant membranes (DRMs) have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. Flotillins-1 and -2 have recently been shown to be large components of erythrocyte DRMs. In this study, we show that a Plasmodium falciparum infection disrupts the association of flotillins with erythrocyte DRMs. Flotillins are probably released from erythrocyte DRMs through the reduction of cholesterol and sphingomyelin levels during the course of a P. falciparum-infection. Although it is well known that a P. falciparum infection can modify the host erythrocyte membrane, this is the first report that P. falciparum can alter the DRM components of erythrocyte membranes.     

Enzymatic properties of the lactate dehydrogenase enzyme from Plasmodium falciparum

The lactate dehydrogenase enzyme from Plasmodium falciparum (PfLDH) is a target for antimalarial compounds owing to structural and functional differences from the human isozymes. The plasmodial enzyme possesses a five-residue insertion in the substrate-specificity loop and exhibits less marked substrate inhibition than its mammalian counterparts. Here we provide a comprehensive kinetic analysis of the enzyme by steady-state and transient kinetic methods. The mechanism deduced by product inhibition studies proves that PfLDH shares a common mechanism with the human LDHs, that of an ordered sequential bireactant system with coenzyme binding first. Transient kinetic analysis reveals that the major rate-limiting step is the closure of the substrate-specificity loop prior to hydride transfer, in line with other LDHs. The five-residue insertion in this loop markedly increases substrate specificity compared with the human muscle and heart isoforms.     

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5-11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.     

Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.     

The genome of Plasmodium falciparum. I: DNA base composition.

Some structural properties of the DNA of Plasmodium falciparum were studied thoroughly using several techniques. Its G+C content was found to be extremely low (17-19%), the lowest reported for a living organism. The DNA seems to be composed only of the four major bases as no methylated bases were detected. This DNA had a Tm value of 62.5 degrees C and its denaturation profile showed no marked intramolecular heterogeneity.