Plasmodium Genome Research Articles

Structures of plasmepsin II from Plasmodium falciparum in complex with two hydroxyethylamine-based inhibitors.

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome of Plasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence of P. falciparum strains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII-NU655 complex. Structural analysis of the PMII-PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275-Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII-NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1' position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-γ(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.

Genome wide in silico analysis of Plasmodium falciparum phosphatome.

BackgroundEukaryotic cellular machineries are intricately regulated by several molecular mechanisms involving transcriptional control, post-translational control and post-translational modifications of proteins (PTMs). Reversible protein phosphorylation/dephosphorylation process, which involves kinases as well as phosphatases, represents an important regulatory mechanism for diverse pathways and systems in all organisms including human malaria parasite, Plasmodium falciparum. Earlier analysis on P. falciparum protein-phosphatome revealed presence of 34 phosphatases in Plasmodium genome. Recently, we re-analysed P. falciparum phosphatome aimed at identifying parasite specific phosphatases.ResultsPlasmodium database (PlasmoDB 9.2) search, combined with PFAM and CDD searches, revealed 67 candidate phosphatases in P. falciparum. While this number is far less than the number of phosphatases present in Homo sapiens, it is almost the same as in other Plasmodium species. These Plasmodium phosphatase proteins were classified into 13 super families based on NCBI CDD search. Analysis of proteins expression profiles of the 67 phosphatases revealed that 44 phosphatases are expressed in both schizont as well as gametocytes stages. Fourteen phosphatases are common in schizont, ring and trophozoite stages, four phosphatases are restricted to gametocytes, whereas another three restricted to schizont stage. The phylogenetic trees for each of the known phosphatase super families reveal a considerable phylogenetic closeness amongst apicomplexan organisms and a considerable phylogenetic distance with other eukaryotic model organisms included in the study. The GO assignments and predicted interaction partners of the parasite phosphatases indicate its important role in diverse cellular processes.ConclusionIn the study presented here, we reviewed the P. falciparum phosphatome to show presence of 67 candidate phosphatases in P. falciparum genomes/proteomes. Intriguingly, amongst these phosphatases, we could identify six Plasmodium specific phosphatases and 33 putative phosphatases that do not have human orthologs, thereby suggesting that these phosphatases have the potential to be explored as novel antimalarial drug targets.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1024) contains supplementary material, which is available to authorized users.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N5, N10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in α-ketoacid dehydrogenase reactions.

Genomes of chimpanzee malaria parasites reveal how they might adapt to humans

Genomes of chimpanzee malaria parasites reveal how they might adapt to humans

Efficient editing of malaria parasite genome using the CRISPR/Cas9 system.

ABSTRACTMalaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites.

Plasmodium genome in blood donors at risk for malaria after several years of residence in Italy

At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy. Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 10(3) -8.5 × 10(4) copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.

PCR targeting Plasmodium mitochondrial genome of DNA extracted from dried blood on filter paper compared to whole blood

BackgroundMonitoring mortality and morbidity attributable to malaria is paramount to achieve elimination of malaria. Diagnosis of malaria is challenging and PCR is a reliable method for identifying malaria with high sensitivity. However, blood specimen collection and transport can be challenging and obtaining dried blood spots (DBS) on filter paper by finger-prick may have advantages over collecting whole blood by venepuncture.MethodsDBS and whole blood were collected from febrile children admitted at the general paediatric wards at a referral hospital in Dar es Salaam, Tanzania. DNA extracted from whole blood and from DBS was tested with a genus-specific PCR targeting the mitochondrial Plasmodium genome. Positive samples by PCR of DNA from whole blood were tested with species-specific PCR targeting the 18S rRNA locus, or sequencing if species-specific PCR was negative. Rapid diagnostic test (RDT) and thin blood smear microscopy was carried out on all patients where remnant whole blood and a blood slide, respectively, were available.ResultsPositivity of PCR was 24.5 (78/319) and 11.2% (52/442) by whole blood and DBS, respectively. All samples positive on DBS were also positive on Plasmodium falciparum species-specific PCR. All RDT positive cases were also positive by DBS PCR. All but three cases with positive blood slides were also positive by DBS.ConclusionsIn this study, PCR for malaria mitochondrial DNA extracted from whole blood was more sensitive than from DBS. However, DBS are a practical alternative to whole blood and detected approximately the same number of cases as RDTs and, therefore, remain relevant for research purposes.

A lysine‐rich membrane‐associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum ‐infected red blood cells

The genomes of malaria parasites (Plasmodium spp.) contain a family of genes encoding proteins with a Plasmodium helical interspersed subtelomeric (PHIST) domain, most of which are predicted to be exported into the parasite-infected human red blood cell (iRBC). Here, using transgenic parasites and a combination of cellular, biochemical, and biophysical assays, we have characterized and determined the function of a novel member of the PHIST protein family in Plasmodium falciparum, termed lysine-rich membrane-associated PHISTb (LyMP). LyMP was shown to associate directly with the cytoskeleton of iRBCs where it plays a role in their abnormal ability to adhere to a protein expressed on vascular endothelial cells, resulting in sequestration. Deletion of LyMP dramatically reduced adhesion of iRBCs to CD36 by 55%, which was completely restored to wild-type levels on complementation. Intriguingly, in the absence of LyMP, formation of RBC membrane knobs and the level of surface exposure of the parasites' major cytoadhesive ligand, PfEMP1, were identical to those for the parental parasite line, demonstrating for the first time an additional mechanism that enhances cytoadherence of iRBCs beyond those already recognized. Our findings identify LyMP as a previously unknown RBC cytoskeletal-binding protein that is likely to be of major significance in the complex pathophysiology of falciparum malaria.-Proellocks, N. I., Herrmann, S., Buckingham, D. W., Hanssen, E., Hodges, E. K., Elsworth, B., Morahan, B. J., Coppel, R. L., Cooke, B. M. A lysine-rich membrane-associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum infected red blood cells.

Molecular cloning, characterization and expression profile of a glutathione peroxidase-like thioredoxin peroxidase (TPxGl) of the rodent malaria parasite Plasmodium berghei

Glutathione peroxidases (GPx) comprise an important group of redox active proteins with diverse functions, including antioxidant defense and signaling. Although the genome of the malaria parasite Plasmodium does not contain a genuine GPx gene a glutathione peroxidase-like thioredoxin peroxidase (TPxGl) has recently been identified and biochemically characterized in the human malaria parasite P. falciparum. To gain more insight into the potential biological function of this enzyme we have cloned and expressed TPxGl of the rodent model system P. berghei (PbTPxGl). Biochemical characterization confirmed that the protein is redox active with the P. berghei thioredoxin system. We compared PbTPxGl to recently characterized thioredoxin-dependent GPx-type proteins of other organisms, and generated the first hypothetical 3D model of a Plasmodium TPxGl, which shows the conservation of the thioredoxin-fold as well as the spatial orientation of a classic GPx catalytic tetrad. In vivo studies indicate that PbTPxGl is continuously expressed in all P. berghei asexual blood stages, gametocytes and in early mosquito-stage parasites. Confocal microscopy suggest a cytoplasmic localization of PbTPxGl in all investigated parasite life stages, specifically in mature ookinetes. Our data provides new insights into the structure and ubiquitous expression of Plasmodium TPxGl and warrants further investigation into this potentially important redox enzyme.

Recurrent bottlenecks in the malaria life cycle obscure signals of positive selection

Detecting signals of selection in the genome of malaria parasites is a key to identify targets for drug and vaccine development. Malaria parasites have a unique life cycle alternating between vector and host organism with a population bottleneck at each transition. These recurrent bottlenecks could influence the patterns of genetic diversity and the power of existing population genetic tools to identify sites under positive selection. We therefore simulated the site-frequency spectrum of a beneficial mutant allele through time under the malaria life cycle. We investigated the power of current population genetic methods to detect positive selection based on the site-frequency spectrum as well as temporal changes in allele frequency. We found that a within-host selective advantage is difficult to detect using these methods. Although a between-host transmission advantage could be detected, the power is decreased when compared with the classical Wright-Fisher (WF) population model. Using an adjusted null site-frequency spectrum that takes the malaria life cycle into account, the power of tests based on the site-frequency spectrum to detect positive selection is greatly improved. Our study demonstrates the importance of considering the life cycle in genetic analysis, especially in parasites with complex life cycles.

Enzymes involved in plastid‐targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver‐stage development

Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver-stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic acid synthesis. Our research shows that apicoplast-targeted Plasmodium yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver-stage development and deletion of the encoding genes resulted in late liver-stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver-stage maturation.

The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.

Using Genetic Methods To Define the Targets of Compounds with Antimalarial Activity

Although phenotypic cellular screening has been used to drive antimalarial drug discovery in recent years, in some cases target-based drug discovery remains more attractive. This is especially true when appropriate high-throughput cellular assays are lacking, as is the case for drug discovery efforts that aim to provide a replacement for primaquine (4-N-(6-methoxyquinolin-8-yl)pentane-1,4-diamine), the only drug that can block Plasmodium transmission to Anopheles mosquitoes and eliminate liver-stage hypnozoites. At present, however, there are no known chemically validated parasite protein targets that are important in all Plasmodium parasite developmental stages and that can be used in traditional biochemical compound screens. We propose that a plethora of novel, chemically validated, cross-stage antimalarial targets still remain to be discovered from the ~5,500 proteins encoded by the Plasmodium genomes. Here we discuss how in vitro evolution of drug-resistant strains of Plasmodium falciparum and subsequent whole-genome analysis can be used to find the targets of some of the many compounds discovered in whole-cell phenotypic screens.

Identification of piggyBac-mediated insertions in Plasmodium berghei by next generation sequencing

BackgroundThe piggyBac transposon system provides a powerful forward genetics tool to study gene function in Plasmodium parasites via random insertion mutagenesis and phenotypic screening. The identification of genotype of piggyBac mutants in the Plasmodium genome is thus an indispensable step in forward genetic analysis. Several PCR-based approaches have been used to identify the piggyBac insertion sites in Plasmodium falciparum and Plasmodium berghei, but all are tedious and inefficient. Next generation sequencing can produce large amounts of sequence data and is particularly suitable for genome-wide association studies. In this study, the Next generation sequencing technology was employed to efficiently identify piggyBac insertion sites in the genome of P. berghei.MethodsPlasmodium berghei parasites were co-transfected with piggyBac donor and helper plasmids. Initially, the classical inverse PCR method was used to identify the existence of piggyBac insertions in the P. berghei genome. The whole genome of post-transfection parasites was subsequently sequenced with a PCR-free paired-end module using the Illumina HiSeq sequencing system. The two distinct methods (‘BLAST method’ and ‘SOAP method’) were employed to identify piggyBac insertion sites in the P. berghei genome with Illumina sequencing data. All the identified piggyBac insertions were further tested by half-nested PCR.ResultsThe inverse PCR method resulted in a very low yield of ten individual insertions identified. Conversely, 47 piggyBac insertions were identified from about 1 Gb of Illumina sequencing data via the two distinct analysis methods. The majority of identified piggyBac insertions were confirmed by half-nested PCR. In addition, 1,850 single nucleotide polymorphisms were identified through alignment of the Illumina sequencing data of the P. berghei ANKA strain used in this study with the reference genome sequences.ConclusionThis study demonstrates that a high-throughput genome sequencing approach is an efficient tool for the identification of piggyBac-mediated insertions in Plasmodium parasites.

Insight into structural and biochemical determinants of substrate specificity of PFI1625c: Correlation analysis of protein-peptide molecular models

Bioinformatics and sequence comparison indicate PFI1625c as a putative metalloprotease present in plasmodium genome. The structure of PFI1625c consists of two domains with nearly identical folding topology. The active site of PFI1625c is located in a large central cavity between the two domains. Substrate binding regions of PFI1625c are lined by E-136, D-140 which provides negatively charged patches whereas F-53 facilitates binding of bulky hydrophobic residues of substrates. Probing PFI1625c active site with 199 different peptides from a combinatorial peptide library indicates preference of PFI1626c toward hydrophobic residue substituted peptides. Correlation analysis of each position of the peptide indicates that Ser 2 is the most crucial residue and no significant improvement was observed until it is mutated to a hydrophobic residue. The peptide P550 (LVIVAKRA) exhibits significantly better interaction within the active site than a template peptide (LSRVAKRA). The molecular dynamic's simulation studies confirms integrity of the complex, with all structures well within the qualitative limit of compactness and stability during the simulation time. There are structural and biochemical differences between PFI1625c with human metalloprotease and these are sufficient enough to allow us to exploit PFI1625c as drug targets. These computationally obtained insights provided clues about substrate selectivity in PFI1625c and it can be used to exploit PFI1625c as a target for future anti-malarial development.

Expression profile of the Plasmodium falciparum intra-erythrocytic stage protein, PF3D7_1363700

BackgroundEfforts to control malaria are demanding due to drug-resistant parasites, insecticide-resistant mosquitoes and poor health infrastructure in malaria-endemic countries. Therefore, the research and development of additional malaria control methods are crucial. For host-parasite interactions, surface antigens and secreted proteins are likely to be involved in infectivity and invasion of host tissues and therefore can be effective targets for control by vaccines, drug therapy, or novel mosquito control methods. In an effort to identify and characterize genes that may have a role in host-parasite interaction, this study describes the expression profile of Plasmodium falciparum PF3D7_1363700.MethodsA P. falciparum gene, PF3D7_1363700, was identified by a search of the annotated Plasmodium genome database. Protein alignments of PF3D7_1363700 orthologues from various Plasmodium species were performed to demonstrate protein similarity. Transcript expression profiles of PF3D7_1363700 were determined via reverse-transcriptase PCR and protein expression was investigated by immunofluorescence assays, western blot analysis and green fluorescent trafficking studies.ResultsThe PF3D7_1363700 protein demonstrates significant similarity with orthologues in other Plasmodium species and appears to be unique to Apicomplexans. The PF3D7_1363700 transcription profile demonstrated expression during the intra-erythrocytic, oocyst sporozoite, and salivary gland sporozoite stages while the PF3D7_1363700 protein was only detected during the intra-erythrocytic stages.ConclusionsThis research utilized an in silico approach to identify a well-conserved protein known as PF3D7_1363700. By molecular, biochemical and cellular analyses, PF3D7_1363700 was discovered to be an intra-erythrocytic-specific stage protein that is unique to Apicomplexans.

H2A.Z/H2B.Z double‐variant nucleosomes inhabit the AT‐rich promoter regions of the Plasmodium falciparum genome

Histone variants are key components of the epigenetic code and evolved to perform specific functions in transcriptional regulation, DNA repair, chromosome segregation and other fundamental processes. Although variants for histone H2A and H3 are found throughout the eukaryotic kingdom, variants of histone H2B and H4 are rarely encountered. H2B.Z is one of those rare H2B variants and is apicomplexan-specific. Here we show that in Plasmodium falciparum H2B.Z localizes to euchromatic intergenic regions throughout intraerythrocytic development and together with H2A.Z forms a double-variant nucleosome subtype. These nucleosomes are enriched in promoters over 3′ intergenic regions and their occupancy generally correlates with the strength of the promoter, but not with its temporal activity. Remarkably, H2B.Z occupancy levels exhibit a clear correlation with the base-composition of the underlying DNA, raising the intriguing possibility that the extreme AT content of the intergenic regions within the Plasmodium genome might be instructive for histone variant deposition. In summary, our data show that the H2A.Z/H2B.Z double-variant nucleosome demarcates putative regulatory regions of the P. falciparum epigenome and likely provides a scaffold for dynamic regulation of gene expression in this deadly human pathogen.