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MBio Oct 2020UDP--acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is...
UDP--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 - and -linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate -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 parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from , an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for 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. 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 -acetyltransferase (GNA1), required for the biosynthesis of UDP--acetylglucosamine (UDP-GlcNAc), is essential for 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 , 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.
Topics: Amino Acid Sequence; Binding Sites; Biosynthetic Pathways; Cryptosporidium parvum; Crystallography, X-Ray; Erythrocytes; Genetic Engineering; Glucosamine 6-Phosphate N-Acetyltransferase; Humans; Malaria, Falciparum; Plasmodium falciparum
PubMed: 33082260
DOI: 10.1128/mBio.02045-20 -
Current Opinion in Microbiology Feb 2023The apicoplast of Plasmodium falciparum is the only source of essential isoprenoid precursors and Coenzyme A (CoA) in the parasite. Isoprenoid precursor synthesis relies... (Review)
Review
The apicoplast of Plasmodium falciparum is the only source of essential isoprenoid precursors and Coenzyme A (CoA) in the parasite. Isoprenoid precursor synthesis relies on the iron-sulfur cluster (FeS) cofactors produced within the apicoplast, rendering FeS synthesis an essential function of this organelle. Recent reports provide important insights into the roles of FeS cofactors and the use of isoprenoid precursors and CoA both inside and outside the apicoplast. Here, we review the recent insights into the roles of these metabolites in blood-stage malaria parasites and discuss new questions that have been raised in light of these discoveries.
Topics: Animals; Humans; Apicoplasts; Parasites; Malaria; Plasmodium falciparum; Terpenes; Protozoan Proteins
PubMed: 36563485
DOI: 10.1016/j.mib.2022.102255 -
Tropical Biomedicine Sep 2020Malaria is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. Plasmodium falciparum (P.... (Review)
Review
Malaria is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. Plasmodium falciparum (P. falciparum), the most virulent malaria parasite in humans, was recently reported to develop resistance against the final efficient antimalarial drug, artemisinin. Little is known about the resistance mechanisms, which further complicates the problem as a proper counteraction is unable to be taken. Hence, the understanding of drug mode of action and its molecular target is valuable knowledge that needs to be considered to develop the next generation of antimalarial drugs. P. falciparum protein kinase (Pf PK) is an attractive target for antimalarial chemotherapy due to its vital roles in all P. falciparum life stages. Moreover, overall structural differences and the presence of unique Pf PKs that are absent in human kinome, suggesting specific inhibition of Pf PK without affecting human cells is achievable. To date, at least 86 eukaryotic protein kinases have been identified in P. falciparum kinome, by which less than 40 were validated as potential targets at the erythrocytes stage. In this review, recent progress of the furthest validated Pf PKs; Pf Nek-1, Pf CDPK1, Pf CDPK4, Pf PKG, and Pf CLK-3 will be briefly discussed.
Topics: Antimalarials; Humans; Malaria, Falciparum; Plasmodium falciparum; Protein Kinase Inhibitors; Protein Kinases
PubMed: 33612795
DOI: 10.47665/tb.37.3.822 -
Nature Communications Feb 2024Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in...
Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.
Topics: Humans; RNA; Plasmodium falciparum; Proteome; RNA-Binding Proteins; Plasmodium
PubMed: 38355719
DOI: 10.1038/s41467-024-45519-1 -
Scientific Reports Nov 2021Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum...
Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.
Topics: Antigens, Protozoan; Child, Preschool; Democratic Republic of the Congo; Diagnostic Tests, Routine; Female; Gene Deletion; Humans; Infant; Malaria, Falciparum; Male; Plasmodium falciparum; Protozoan Proteins; Time Factors
PubMed: 34837020
DOI: 10.1038/s41598-021-02452-3 -
Malaria Journal Oct 2021The emergence and spread of malaria drug resistance have resulted in the need to understand disease mechanisms and importantly identify essential targets and potential...
BACKGROUND
The emergence and spread of malaria drug resistance have resulted in the need to understand disease mechanisms and importantly identify essential targets and potential drug candidates. Malaria infection involves the complex interaction between the host and pathogen, thus, functional interactions between human and Plasmodium falciparum is essential to obtain a holistic view of the genetic architecture of malaria. Several functional interaction studies have extended the understanding of malaria disease and integrating such datasets would provide further insights towards understanding drug resistance and/or genetic resistance/susceptibility, disease pathogenesis, and drug discovery.
METHODS
This study curated and analysed data including pathogen and host selective genes, host and pathogen protein sequence data, protein-protein interaction datasets, and drug data from literature and databases to perform human-host and P. falciparum network-based analysis. An integrative computational framework is presented that was developed and found to be reasonably accurate based on various evaluations, applications, and experimental evidence of outputs produced, from data-driven analysis.
RESULTS
This approach revealed 8 hub protein targets essential for parasite and human host-directed malaria drug therapy. In a semantic similarity approach, 26 potential repurposable drugs involved in regulating host immune response to inflammatory-driven disorders and/or inhibiting residual malaria infection that can be appropriated for malaria treatment. Further analysis of host-pathogen network shortest paths enabled the prediction of immune-related biological processes and pathways subverted by P. falciparum to increase its within-host survival.
CONCLUSIONS
Host-pathogen network analysis reveals potential drug targets and biological processes and pathways subverted by P. falciparum to enhance its within malaria host survival. The results presented have implications for drug discovery and will inform experimental studies.
Topics: Antimalarials; Computer Simulation; Drug Discovery; Drug Resistance; Humans; Malaria, Falciparum; Plasmodium falciparum; Protein Interaction Mapping; Protozoan Proteins
PubMed: 34702263
DOI: 10.1186/s12936-021-03955-0 -
Cell Reports Apr 2023Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of...
Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of this protein is not yet established. Here, we report an apurinic/apyrimidinic site-driven intrinsic nuclease activity of PfAlba3 assisted by divalent metal ions. Surface plasmon resonance and atomic force microscopy confirm sequence non-specific DNA binding by PfAlba3. Upon binding, PfAlba3 cleaves double-stranded DNA (dsDNA) hydrolytically. Mutational studies coupled with mass spectrometric analysis indicate that K23 is the essential residue in modulating the binding to DNA through acetylation-deacetylation. We further demonstrate that PfSir2a interacts and deacetylates K23-acetylated PfAlba3 in favoring DNA binding. Hence, K23 serves as a putative molecular switch regulating the nuclease activity of PfAlba3. Thus, the nuclease activity of PfAlba3, along with its apurinic/apyrimidinic (AP) endonuclease feature identified in this study, indicates a role of PfAlba3 in DNA-damage response that may have a far-reaching consequence in Plasmodium pathogenicity.
Topics: Plasmodium falciparum; Protein Binding; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA; DNA Repair
PubMed: 36947546
DOI: 10.1016/j.celrep.2023.112292 -
MBio Jan 2020New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured , against fresh Ugandan isolates, and in murine malaria models....
New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured , against fresh Ugandan isolates, and in murine malaria models. To gain mechanistic insights, we selected for isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC) increased from 18-118 nM to 180-890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3. Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs.
Topics: Antimalarials; Chromatography, Liquid; DNA Mutational Analysis; Drug Resistance; Humans; Malaria, Falciparum; Mass Spectrometry; Molecular Structure; Mutation; Plasmodium falciparum; Polymorphism, Single Nucleotide; Protozoan Proteins; Sumoylation; Ubiquitination
PubMed: 31992618
DOI: 10.1128/mBio.02640-19 -
Parasitology Research Jun 2020RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure....
RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7_0110700 and PF3D7_1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 β-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7_0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology.
Topics: Amino Acid Sequence; Cell Nucleus; Chromatin; Gene Expression; Histone Chaperones; Histones; Life Cycle Stages; Plasmodium falciparum; Protein Binding; Protein Conformation; Protozoan Proteins; Recombinant Fusion Proteins
PubMed: 32363442
DOI: 10.1007/s00436-020-06669-5 -
Scientific Reports Feb 2021Cytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan...
Cytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan specific protein appears to be a crucial factor in maintaining stability of the parasite cytoskeletal assemblies. However, the function of apicortin, in terms of interaction with microtubules still remains elusive. Herein, we have attempted to elucidate the function of Plasmodium falciparum apicortin by monitoring its interaction with two main components of parasite microtubular structure, α-tubulin-I and β-tubulin through in silico and in vitro studies. Further, a p25 domain binding generic drug Tamoxifen (TMX), was used to disrupt PfApicortin-tubulin interactions which led to the inhibition in growth and progression of blood stage life cycle of P. falciparum.
Topics: Adaptor Proteins, Signal Transducing; Microtubules; Plasmodium falciparum; Protein Binding; Protozoan Proteins; Tubulin
PubMed: 33633135
DOI: 10.1038/s41598-021-83513-5