Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.339
Filtrar
1.
Elife ; 112022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36097817

RESUMO

Plasmodium falciparum, the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of PfA-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.


Malaria is a disease spread by mosquitoes. When infected insects bite the skin, they inject parasites called Plasmodium into the host. The symptoms of the disease then develop when Plasmodium infect host red blood cells. These parasites cannot make the raw materials to build their own proteins, so instead, they digest haemoglobin ­ the protein used by red blood cells to carry oxygen ­ and use its building blocks to produce proteins. Blocking the digestion of haemoglobin can stop malaria infections in their tracks, but it is unclear how exactly Plasmodium parasites break down the protein. Researchers think that a group of four enzymes called aminopeptidases are responsible for the final stage in this digestion, releasing the amino acids that make up haemoglobin. However, the individual roles of each of these aminopeptidases are not yet known. To start filling this gap, Edgar et al. set out to study one of these aminopeptidases, called PfA-M17. First, they genetically modified Plasmodium falciparum parasites so that the levels of this aminopeptidase were reduced during infection. Without the enzyme, the parasites were unable to grow. The next step was to confirm that this was because PfA-M17 breaks down haemoglobin, and not for another reason. To test this, Edgar et al. designed a new molecule that could stop PfA-M17 from releasing amino acids. This molecule, which they called 'compound 3', had the same effect as reducing the levels of PfA-M17. Further analysis showed that the amino acids that PfA- M17 releases match the amino acids found in haemoglobin. Malaria causes hundreds of thousands of deaths per year. Although there are treatments available, the Plasmodium parasites are starting to develop resistance. Confirming the role of PfA-M17 provides a starting point for new studies by parasitologists, biologists, and drug developers. This could lead to the development of chemicals that block this enzyme, forming the basis for new treatments.


Assuntos
Malária Falciparum , Plasmodium falciparum , Aminopeptidases/química , Aminopeptidases/genética , Digestão , Hemoglobinas , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Inibidores de Proteases , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
2.
Trends Parasitol ; 38(10): 868-881, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35999149

RESUMO

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp.


Assuntos
Apicomplexa , Apicoplastos , Toxoplasma , Apicomplexa/genética , Apicomplexa/metabolismo , Apicoplastos/genética , Elétrons , Ferredoxinas/genética , Ferredoxinas/metabolismo , Ferro/metabolismo , NADP/metabolismo , Oxirredução , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , RNA de Transferência/metabolismo , Enxofre/metabolismo , Terpenos/metabolismo , Toxoplasma/genética
3.
Hemoglobin ; 46(2): 100-105, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35924733

RESUMO

Understanding the key regulator of iron homeostasis is critical to the improvement of iron supplementation practices in malaria-endemic areas. This study aimed to determine iron indices and hepcidin (HEPC) level in patients infected with Plasmodium falciparum compared to apparently healthy, malaria-negative subjects in Hodeidah, Yemen. The study included 70 Plasmodium falciparum-infected and 20 malaria-negative adults. Blood films were examined for detection and estimation of parasitemia. Hemoglobin (Hb) level was measured using an automated hematology analyzer. Serum iron and total iron binding capacity (TIBC) were determined by spectrophotometric methods. Levels of serum ferritin (FER) and HEPC were measured by enzyme-linked immunosorbent assays. Data were stratified by sex and age. Comparable Hb levels were found in P. falciparum-infected patients and malaria-negative subjects in each sex and age group (p > 0.05). Compared to their malaria-negative counterparts, disturbed iron homeostasis in patients was evidenced by the significantly lower serum iron levels in females (p = 0.007) and those aged <25 years (p = 0.02) and the significantly higher TIBC in males (p = 0.008). Levels of serum FER and HEPC were significantly elevated in P. falciparum-infected patients compared to the corresponding malaria-negative participants (p < 0.001). Serum FER correlated positively with parasite density (p = 0.004). In conclusion, patients with uncomplicated P. falciparum in Hodeidah display elevated levels of serum HEPC and FER. Hemoglobin level may not reflect the disturbed iron homeostasis in these patients. The combined measurement of iron indices and HEPC provides comprehensive information on the iron status so that the right intervention can be chosen.


Assuntos
Malária Falciparum , Malária , Adulto , Feminino , Ferritinas , Hemoglobinas/metabolismo , Hepcidinas , Humanos , Ferro/metabolismo , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Masculino , Plasmodium falciparum/metabolismo , Iêmen/epidemiologia
4.
PLoS One ; 17(8): e0273357, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35984838

RESUMO

Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.


Assuntos
Antimaláricos , Malária Falciparum , Malária , Antimaláricos/uso terapêutico , Atovaquona/uso terapêutico , Biotinilação , Combinação de Medicamentos , Humanos , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proguanil/uso terapêutico , Proteômica
5.
Parasitol Int ; 91: 102648, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35988900

RESUMO

Rapid diagnostic tests (RDTs) based on immunochromatographic detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) have been frequently used for malaria diagnosis. The HRP2-based RDTs are highly sensitive and easy to use; however, their sensitivity may be low in detecting P. falciparum strains carrying deletion of the pfhrp2 and pfhrp3 genes encoding HRP2 and HRP3, respectively. The automated hematology analyzer XN-31, developed by Sysmex (Kobe, Japan) to aid in malaria diagnosis, has higher sensitivity than RDTs owing to a unique automated nucleic acid staining technology that has shown great potential in clinical settings. In this study, we compared the performance of the XN-31 analyzer and two RDTs to detect pfhrp2- and/or pfhrp3-deleted parasites cultured in vitro. The analyses showed that the analyzer was not only as sensitive to pfhrp2- and/or pfhrp3-deleted strains as it was to the wild-type strain but also had higher sensitivity than the RDTs. These results suggested that the XN-31 analyzer is useful for rapid and reliable detection of pfhrp2- and/or pfhrp3-deleted parasites in clinical settings.


Assuntos
Hematologia , Malária Falciparum , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Histidina/metabolismo , Humanos , Japão , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
6.
Nat Commun ; 13(1): 4537, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927261

RESUMO

The malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and then ruptures the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also autoprocesses to generate different isoforms. The function of this processing is not understood. We have mapped the self-cleavage sites and have constructed parasites with cleavage site mutations. Surprisingly, a quadruple mutant that remains full-length retains in vitro activity, is trafficked normally, and supports normal egress, invasion and parasite growth. The N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.


Assuntos
Malária Falciparum , Plasmodium falciparum , Ácido Aspártico Endopeptidases , Eritrócitos/parasitologia , Humanos , Malária Falciparum/parasitologia , Peptídeo Hidrolases/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
7.
Parasit Vectors ; 15(1): 309, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36042490

RESUMO

Malaria is a life-threatening parasitic disease caused by members of the genus Plasmodium. The development and spread of drug-resistant strains of Plasmodium parasites represent a major challenge to malaria control and elimination programmes. Evaluating genetic polymorphism in a drug target improves our understanding of drug resistance and facilitates drug design. Approximately 450 and 19 whole-genome assemblies of Plasmodium falciparum and Plasmodium vivax, respectively, are currently available, and numerous sequence variations have been found due to the presence of single nucleotide polymorphism (SNP). In the study reported here, we analysed global SNPs in the malaria parasite aminoacyl-tRNA synthetases (aaRSs). Our analysis revealed 3182 unique SNPs in the 20 cytoplasmic P. falciparum aaRSs. Structural mapping of SNPs onto the three-dimensional inhibitor-bound complexes of the three advanced drug targets within aaRSs revealed a remarkably low mutation frequency in the crucial aminoacylation domains, low overall occurrence of mutations across samples and high conservation in drug/substrate binding regions. In contrast to aaRSs, dihydropteroate synthase (DHPS), also a malaria drug target, showed high occurrences of drug resistance-causing mutations. Our results show that it is pivotal to screen potent malaria drug targets against global SNP profiles to assess genetic variances to ensure success in designing drugs against validated targets and tackle drug resistance early on.


Assuntos
Aminoacil-tRNA Sintetases , Antimaláricos , Malária Falciparum , Malária , Parasitos , Plasmodium , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Animais , Antimaláricos/farmacologia , Di-Hidropteroato Sintase/genética , Resistência a Medicamentos/genética , Genômica , Malária Falciparum/parasitologia , Plasmodium/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/metabolismo
8.
PLoS Pathog ; 18(8): e1009882, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35930605

RESUMO

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins the parasite's pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer's cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer's clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, identified only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking.


Assuntos
Plasmodium falciparum , Proteínas de Protozoários , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Organelas/metabolismo , Plasmodium falciparum/metabolismo , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
9.
ACS Infect Dis ; 8(9): 1962-1974, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36037410

RESUMO

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.


Assuntos
Antimaláricos , Lisina-tRNA Ligase , Malária , Antimaláricos/química , Antimaláricos/farmacologia , Descoberta de Drogas , Humanos , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/metabolismo , Plasmodium falciparum/metabolismo
10.
Curr Opin Microbiol ; 69: 102196, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36037636

RESUMO

Most eukaryotic proteins undergo post-translational modifications (PTMs) that significantly alter protein properties, regulate diverse cellular processes and increase proteome complexity. Among these PTMs, lipidation plays a unique and key role in subcellular trafficking, signalling and membrane association of proteins through altering substrate function, and hydrophobicity via the addition and removal of lipid groups. Three prevalent classes of lipid modifications in Plasmodium parasites include prenylation, myristoylation, and palmitoylation that are important for regulating parasite-specific molecular processes. The enzymes that catalyse these lipid attachments have also been explored as potential drug targets for antimalarial development. In this review, we discuss these lipidation processes in Plasmodium spp. and the methodologies that have been used to identify these modifications in the deadliest species of malaria parasite, Plasmodium falciparum. We also discuss the development status of inhibitors that block these pathways.


Assuntos
Parasitos , Plasmodium , Animais , Lipídeos , Plasmodium/genética , Plasmodium/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo
11.
Biochim Biophys Acta Proteins Proteom ; 1870(9): 140832, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35934300

RESUMO

Most antimalarial therapeutics, including chloroquine and artemisinin, induce free heme-mediated toxicity in Plasmodium. This cytotoxic heme is produced as a by-product during the large-scale digestion of host hemoglobin. Conversion of this host-derived heme into inert crystalline hemozoin is the only defense mechanism in Plasmodium against heme-induced cytotoxicity. Heme detoxification protein (HDP), a highly conserved plasmodial protein, is reported to be the most efficient biological mediator for heme to hemozoin transformation. Despite its significance, HDP has never been extensively studied for heme transformation into hemozoin. Therefore, we wish to develop a method to study the HDP-mediated transformation of heme into hemozoin. We have adopted, modified, and optimized the pyridine hemochrome assay to study HDP catalysis and use substrate and time kinetics to study the HDP-mediated transformation of heme into hemozoin. In contrast to the previously reported assay for HDP, we found that the new assay is more precise, accurate, and handy, making it more suitable for kinetic studies. HDP-mediated transformation of heme into hemozoin is not a single-step process, and involves a transient intermediate, most likely a cyclic heme dimer. The kinetics and the manner of HDP-mediated hemozoin production are dependent on the substrate concentration, and a small fraction of substrate remains untransformed to hemozoin irrespective of reaction time. Combining HDP as a catalyst and the pyridine hemochrome assay will facilitate the efficient screening of future antimalarials.


Assuntos
Antimaláricos , Hemeproteínas , Plasmodium , Antimaláricos/farmacologia , Heme/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Cinética , Plasmodium falciparum/metabolismo
12.
mBio ; 13(4): e0189722, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35938722

RESUMO

Artemisinin resistance in Plasmodium falciparum has been associated with a mutation in the NLI-interacting factor-like phosphatase PfNIF4, in addition to the mutations in the Kelch13 protein as the major determinant. We found that PfNIF4 was predominantly expressed at the schizont stage and localized in the nuclei of the parasite. To elucidate the functions of PfNIF4 in P. falciparum, we performed PfNIF4 knockdown (KD) using the inducible ribozyme system. PfNIF4 KD attenuated merozoite invasion and affected gametocytogenesis. PfNIF4 KD parasites also showed significantly increased in vitro susceptibility to artemisinins. Transcriptomic and proteomic analysis revealed that PfNIF4 KD led to the downregulation of gene categories involved in invasion and artemisinin resistance (e.g., mitochondrial function, membrane, and Kelch13 interactome) at the trophozoite and/or schizont stage. Consistent with PfNIF4 being a protein phosphatase, PfNIF4 KD resulted in an overall upregulation of the phosphoproteome of infected erythrocytes. Quantitative phosphoproteomic profiling identified a set of PfNIF4-regulated phosphoproteins with functional similarity to FCP1 substrates, particularly proteins involved in chromatin organization and transcriptional regulation. Specifically, we observed increased phosphorylation of Ser2/5 of the tandem repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII) upon PfNIF4 KD. Furthermore, using the TurboID-based proteomic approach, we identified that PfNIF4 interacted with the RNAPII components, AP2-domain transcription factors, and chromatin-modifiers and binders. These findings suggest that PfNIF4 may act as the RNAPII CTD phosphatase, regulating the expression of general and parasite-specific cellular pathways during the blood-stage development. IMPORTANCE Protein phosphorylation regulates a multitude of cellular processes. The eukaryotic FCP1 phosphatase acts as a CTD-phosphatase to critically balance the phosphorylation status of the CTD of the RNAPII, controlling the accurate execution of the transcription process. Here, we identified PfNIF4 as the FCP1-like phosphatase in P. falciparum. PfNIF4 KD specifically downregulated genes involved in merozoite invasion, resulting in the attenuation of this process. Consistent with the earlier finding of the association of PfNIF4 mutations with artemisinin resistance in Southeast Asian parasite populations, PfNIF4 KD significantly increased in vitro susceptibility to artemisinins. The regulation of these cellular processes in P. falciparum by PfNIF4 is likely realized through RNAPII-mediated transcription, because PfNIF4 was found to interact with RNAPII subunits and KD of PfNIF4 caused CTD hyperphosphorylation. Our results reveal the functions of the PfNIF4 phosphatase in controlling the transcription of invasion- and resistance-related genes in the malaria parasite.


Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Animais , Antimaláricos/farmacologia , Artemisininas/metabolismo , Artemisininas/farmacologia , Malária Falciparum/parasitologia , Merozoítos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Plasmodium falciparum/metabolismo , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Polimerase II/metabolismo , Esquizontes/genética
13.
Traffic ; 23(9): 442-461, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36040075

RESUMO

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs-modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.


Assuntos
Antimaláricos , Malária Falciparum , Malária , Parasitos , Animais , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Eritrócitos/parasitologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Parasitos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
14.
Nat Commun ; 13(1): 4400, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35906227

RESUMO

Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.


Assuntos
Culicidae , Malária Falciparum , Animais , Culicidae/metabolismo , Glicosilação , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trombospondinas/metabolismo , Triptofano/metabolismo
15.
Mol Pharmacol ; 102(3): 172-182, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35798366

RESUMO

Human and animal malaria parasites increase their host erythrocyte permeability to a broad range of solutes as mediated by parasite-associated ion channels. Molecular and pharmacological studies have implicated an essential role in parasite nutrient acquisition, but inhibitors suitable for development of antimalarial drugs are missing. Here, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and is refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on the plasmodial surface anion channel, a channel linked to parasite-encoded RhopH proteins. These studies identify pyridazinones as novel and tractable antimalarial scaffolds with a defined mechanism of action. SIGNIFICANCE STATEMENT: Because antimalarial drugs are prone to evolving resistance in the virulent human P. falciparum pathogen, new therapies are needed. This study has now developed a novel drug-like series of pyridazinones that target an unexploited parasite anion channel on the host cell surface, display excellent in vitro and in vivo ADME properties, are refractory to acquired resistance, and demonstrate a well defined mechanism of action.


Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Animais , Ânions/química , Ânions/metabolismo , Antimaláricos/farmacologia , Eritrócitos/metabolismo , Humanos , Nutrientes , Plasmodium falciparum/metabolismo
16.
Biochem Pharmacol ; 203: 115154, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35798201

RESUMO

The development of resistance to current antimalarial therapies remains a significant source of concern. To address this risk,newdrugswithnoveltargetsin distinct developmental stages ofPlasmodiumparasites are required. In the current study,we have targetedP. falciparumTubulin(PfTubulin)proteins which represent some of thepotentialdrug targetsfor malaria chemotherapy. PlasmodialMicrotubules (MTs) play a crucial role during parasite proliferation, growth, and transmission, which render them highlydesirabletargets for the development ofnext-generation chemotherapeutics. Towards this,we have evaluated the antimalarial activity ofTubulintargetingcompounds received from theMedicines for Malaria Venture (MMV)"Pathogen Box"against the human malaria parasite,P. falciparumincluding 3D7 (chloroquine and artemisinin sensitive strain), RKL-9 (chloroquine-resistant strain), and R539T (artemisinin-resistant strain). At nanomolar concentrations, the filtered-out compounds exhibitedpronouncedmultistage antimalarialeffects across the parasite life cycle, including intra-erythrocytic blood stages, liver stage parasites, gametocytes, and ookinetes. Concomitantly, these compoundswere found toimpedemale gamete ex-flagellation, thus showingtheir transmission-blocking potential. Target mining of these potent compounds, by combining in silico, biochemical and biophysical assays,implicatedPfTubulinas their moleculartarget, which may possibly act bydisruptingMT assembly dynamics by binding at the interface of α-ßTubulin-dimer.Further, the promising ADME profile of the parent scaffold supported its consideration as a lead compound for further development.Thus, our work highlights the potential of targetingPfTubulin proteins in discovering and developing next-generation, multistage antimalarial agents against Multi-Drug Resistant (MDR) malaria parasites.


Assuntos
Antimaláricos , Artemisininas , Malária , Acesso à Informação , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Cloroquina/farmacologia , Humanos , Malária/tratamento farmacológico , Plasmodium falciparum/metabolismo , Tubulina (Proteína)/metabolismo
17.
Methods Mol Biol ; 2470: 327-342, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35881356

RESUMO

Identification of P. falciparum infected erythrocyte surface ligands (such as PfEMP1) matched with the host receptors they interact with, as well as identification of PfEMP1 domains that are targets of protective immunity, are important for understanding of the pathophysiology of severe malaria (SM) and for design of novel vaccine candidates. In addition, identification of small-molecule drugs that can prevent or reverse receptor-ligand domain interactions could provide new tools for adjunctive therapy in SM. This protocol describes how to prepare functionally intact PfEMP1 proteins in mammalian cells (COS-7) and immobilize them on the surface of BioPlex beads. Furthermore, the protocol described how to identify PfEMP1 constructs that bind to specific host receptors or to immunoglobulins (IgG, IgM, etc.), and how to measure inhibition of the receptor binding to PfEMP1 constructs by small-molecule compounds or serum/plasma.


Assuntos
Malária Falciparum , Plasmodium falciparum , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários/metabolismo , Eritrócitos/metabolismo , Humanos , Ligantes , Mamíferos/metabolismo , Plasmodium falciparum/metabolismo , Polímeros , Proteínas de Protozoários/metabolismo
18.
Biomolecules ; 12(8)2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35892329

RESUMO

Heat shock protein 90 (Hsp90) is one of the major guardians of cellular protein homeostasis, through its specialized molecular chaperone properties. While Hsp90 has been extensively studied in many prokaryotic and higher eukaryotic model organisms, its structural, functional, and biological properties in parasitic protozoans are less well defined. Hsp90 collaborates with a wide range of co-chaperones that fine-tune its protein folding pathway. Co-chaperones play many roles in the regulation of Hsp90, including selective targeting of client proteins, and the modulation of its ATPase activity, conformational changes, and post-translational modifications. Plasmodium falciparum is responsible for the most lethal form of human malaria. The survival of the malaria parasite inside the host and the vector depends on the action of molecular chaperones. The major cytosolic P. falciparum Hsp90 (PfHsp90) is known to play an essential role in the development of the parasite, particularly during the intra-erythrocytic stage in the human host. Although PfHsp90 shares significant sequence and structural similarity with human Hsp90, it has several major structural and functional differences. Furthermore, its co-chaperone network appears to be substantially different to that of the human host, with the potential absence of a key homolog. Indeed, PfHsp90 and its interface with co-chaperones represent potential drug targets for antimalarial drug discovery. In this review, we critically summarize the current understanding of the properties of Hsp90, and the associated co-chaperones of the malaria parasite.


Assuntos
Proteínas de Choque Térmico HSP90 , Malária Falciparum , Chaperonas Moleculares , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Malária Falciparum/parasitologia , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo
19.
Nat Commun ; 13(1): 4067, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35831417

RESUMO

Plasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA, provides the parasite with protection from macrophage phagocytosis and clearance in the humanized mice. Furthermore, parasites adapted to thrive in the humanized mice show reduced NK cell-mediated killing through interaction with the immune inhibitory receptor, LILRB1. Taken together, these findings reveal new insights into the molecular and cellular mechanisms that the parasite utilizes to coordinate immune escape in vivo. Identification and targeting of these specific parasite variant surface antigens crucial for immune evasion provides a unique approach for therapy.


Assuntos
Malária Falciparum , Plasmodium falciparum , Animais , Antígenos de Protozoários , Antígenos de Superfície/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Camundongos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
20.
Methods Mol Biol ; 2470: 19-25, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35881335

RESUMO

The pathogenesis of malaria is largely attributable to the parasite's ability to modulate its cytoadhesion phenotype. This relates to the multigenic families comprising dozens to hundreds of members, whose expression, often mutually exclusive, allows the parasite to vary its adhesive properties and antigenic appearance. This phenomenon is mainly described for the variant surface antigens that the parasite expresses on the infected erythrocyte. In order to decipher these gene expression spectra and identify potential antigenic candidates and/or targets of therapeutic interest, the analysis of the transcriptomes of the parasites directly isolated from patients with well-defined clinical presentation is important. RNA stabilization is an absolute prerequisite for a precise and accurate transcriptome profiling. Immediate stabilization of RNA of biological samples is therefore necessary to prevent degradation by ribonucleases (RNase) or cellular changes. This chapter described methodology for preserving parasite RNA samples from malaria patients in the field for transcriptome studies.


Assuntos
Malária Falciparum , Malária , Parasitos , Animais , Antígenos/metabolismo , Eritrócitos/parasitologia , Malária/parasitologia , Malária Falciparum/parasitologia , Parasitos/genética , Plasmodium falciparum/metabolismo , RNA/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...