scholarly journals Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Jordi Chi ◽  
Marta Cova ◽  
Matilde de las Rivas ◽  
Ana Medina ◽  
Rafael Junqueira Borges ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Apicomplexan Parasite ◽  
Blood Stage ◽  
Host Cells ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Hexosamine Pathway ◽  
Depth Analysis ◽  
Stage Development

ABSTRACT UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria. IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite’s ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

scholarly journals Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay

2013 ◽  
Vol 57 (12) ◽  
pp. 6050-6062 ◽  
Author(s):  
Leonardo Lucantoni ◽  
Sandra Duffy ◽  
Sophie H. Adjalley ◽  
David A. Fidock ◽  
Vicky M. Avery
Keyword(s):  
Plasmodium Falciparum ◽  
High Throughput ◽  
Early Stage ◽  
Blood Stage ◽  
Stage I ◽  
Mosquito Vector ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Malaria Box ◽  
Gametocytocidal Activity

ABSTRACTThe design of new antimalarial combinations to treatPlasmodium falciparuminfections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinantP. falciparumline expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the early-stage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC50s) below 2.5 μM. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of ≤5%, an average signal-to-noise ratio (S:N) of >30, and a Z′ of ∼0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy.

scholarly journals Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite

2010 ◽  
Vol 9 (7) ◽  
pp. 1064-1074 ◽  
Author(s):  
Madhusudan Kadekoppala ◽  
Solabomi A. Ogun ◽  
Steven Howell ◽  
Ruwani S. Gunaratne ◽  
Anthony A. Holder
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Expression ◽  
Surface Protein ◽  
Gene Families ◽  
Merozoite Surface Protein ◽  
Blood Stage ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Specific Proteins ◽  
Proteolytic Cleavages

ABSTRACT Proteins located on Plasmodium falciparum merozoites, the invasive form of the parasite's asexual blood stage, are of considerable interest in vaccine research. Merozoite surface protein 7 (MSP7) forms a complex with MSP1 and is encoded by a member of a multigene family located on chromosome 13. The family codes for MSP7 and five MSP7-related proteins (MSRPs). In the present study, we have investigated the expression and the effect of msrp gene deletion at the asexual blood stage. In addition to msp7, msrp2, msrp3, and msrp5 are transcribed, and mRNA was easily detected by hybridization analysis, whereas mRNA for msrp1 and msrp4 could be detected only by reverse transcription (RT)-PCR. Notwithstanding evidence of transcription, antibodies to recombinant MSRPs failed to detect specific proteins, except for antibodies to MSRP2. Sequential proteolytic cleavages of MSRP2 resulted in 28- and 25-kDa forms. However, MSRP2 was absent from merozoites; the 25-kDa MSRP2 protein (MSRP225) was soluble and secreted upon merozoite egress. The msrp genes were deleted by targeted disruption in the 3D7 line, leading to ablation of full-length transcripts. MSRP deletion mutants had no detectable phenotype, with growth and invasion characteristics comparable to those of the parental parasite; only the deletion of MSP7 led to a detectable growth phenotype. Thus, within this family some of the genes are transcribed at a significant level in asexual blood stages, but the corresponding proteins may or may not be detectable. Interactions of the expressed proteins with the merozoite also differ. These results highlight the potential for unexpected differences of protein expression levels within gene families.

scholarly journals Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

2013 ◽  
Vol 13 (5) ◽  
pp. 550-559 ◽  
Author(s):  
Ben C. L. van Schaijk ◽  
T. R. Santha Kumar ◽  
Martijn W. Vos ◽  
Adam Richman ◽  
Geert-Jan van Gemert ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Plasmodium Falciparum ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Blood Stage ◽  
Acid Biosynthesis ◽  
Asexual Blood Stage ◽  
Liver Stage ◽  
Content Type ◽  
Fas Ii

ABSTRACT The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum , the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F . Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii , where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

scholarly journals CCR4-Associated Factor 1 Coordinates the Expression of Plasmodium falciparum Egress and Invasion Proteins

2011 ◽  
Vol 10 (9) ◽  
pp. 1257-1263 ◽  
Author(s):  
Bharath Balu ◽  
Steven P. Maher ◽  
Alena Pance ◽  
Chitra Chauhan ◽  
Anatoli V. Naumov ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulation Of Gene Expression ◽  
Host Cells ◽  
Asexual Blood Stage ◽  
New Host ◽  
Associated Factor ◽  
Content Type ◽  
Content Development ◽  
Significant Gene ◽  
Parasite Egress

ABSTRACT Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.

scholarly journals Whole-Cell Phenotypic Screening of Medicines for Malaria Venture Pathogen Box Identifies Specific Inhibitors of Plasmodium falciparum Late-Stage Development and Egress

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Alok Tanala Patra ◽  
Tejashri Hingamire ◽  
Meenakshi A. Belekar ◽  
Aoli Xiong ◽  
Gowtham Subramanian ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Late Stage ◽  
Antimalarial Activity ◽  
Target Identification ◽  
Calcium Ionophore ◽  
Cellular Phenotype ◽  
Mechanistic Studies ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Stage Development

ABSTRACT We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum. First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 μM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 μM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 μM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.

scholarly journals The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development

2018 ◽  
Vol 5 (1) ◽  
pp. 32-41 ◽  
Author(s):  
Cletus A. Wezena ◽  
Romy Alisch ◽  
Alexandra Golzmann ◽  
Linda Liedgens ◽  
Verena Staudacher ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Stage ◽  
Asexual Blood Stage ◽  
Stage Development

scholarly journals Organelle Dynamics in Apicomplexan Parasites

mBio ◽  
2021 ◽  
Author(s):  
Julie M. J. Verhoef ◽  
Markus Meissner ◽  
Taco W. A. Kooij
Keyword(s):  
Plasmodium Falciparum ◽  
Toxoplasma Gondii ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Parental Cell ◽  
Liver Stage ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Stage Development

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium falciparum , are the cause of many important human and veterinarian diseases. While T. gondii tachyzoites replicate through endodyogeny, during which two daughter cells are formed within the parental cell, P. falciparum replicates through schizogony, where up to 32 parasites are formed in a single infected red blood cell and even thousands of daughter cells during mosquito- or liver-stage development.

scholarly journals The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ivan Campeotto ◽  
Francis Galaway ◽  
Shahid Mehmood ◽  
Lea K. Barfod ◽  
Doris Quinkert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
Structural Information ◽  
Blood Stage ◽  
Site Directed Mutagenesis ◽  
Effective Vaccine ◽  
Binding Region ◽  
Fab Fragment ◽  
Content Type ◽  
Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

scholarly journals Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

2013 ◽  
Vol 82 (1) ◽  
pp. 152-164 ◽  
Author(s):  
K. Sony Reddy ◽  
Alok K. Pandey ◽  
Hina Singh ◽  
Tajali Sahar ◽  
Amlabu Emmanuel ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Neutralizing Antibodies ◽  
Malaria Vaccine ◽  
Expression System ◽  
Full Length ◽  
Blood Stage ◽  
Parasite Protein ◽  
Content Type ◽  
Erythrocyte Binding ◽  
Blood Stage Malaria

ABSTRACTPlasmodium falciparumreticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number ofP. falciparumstrains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 inEscherichia colithat exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number ofP. falciparumstrains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies.P. falciparumhas the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwideP. falciparumstrains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.

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