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1.
Nature ; 578(7794): 321-325, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996846

RESUMO

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with D-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from D-glucose) are just as critical for transport as the residues that directly coordinate D-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.


Assuntos
Malária/parasitologia , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Açúcares/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Glucose/química , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/química , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
2.
Georgian Med News ; (294): 103-108, 2019 Sep.
Artigo em Russo | MEDLINE | ID: mdl-31687959

RESUMO

The article overviews some issues of the severe course of tropical malaria. In addition to the analysis of the ongoing situation with malaria in Russia, a general clinical picture of the severe course of tropical malaria is discussed. The main part of the overview includes a detailed analysis of current data on the molecular genetic aspects of the erythrocytes' adhesion in the case of tropical malaria. The main elements involved in the process of binding red blood cells and, as a result, in the process of their adhesion to other cells of the human body were considered in detail. Data were studied and summarized not only on protein interactions between an infected red cell and its cellular environment, but also on the genetic characteristics of the parasite leading to similar molecular-biological processes. In addition to the study of protein PfEMP1 role which is nowadays well-considered in the literature, the most up-to-date but less reported data on erythrocyte adhesion proteins STEVOR and RIFIN were also included. The team of authors hopes that this publication will help to get a deeper insight into the problem of erythrocyte adhesion in the course of complicated malaria infection forms and to summarize some of the available data on this issue.


Assuntos
Antígenos de Protozoários/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/parasitologia , Eritrócitos/parasitologia , Malária Falciparum , Proteínas de Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Antígenos de Protozoários/química , Humanos , Malária Falciparum/sangue , Proteínas de Membrana/química , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/química , Federação Russa
3.
Malar J ; 18(1): 342, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590674

RESUMO

BACKGROUND: Chloroquine (CQ) was the drug of choice for decades in the treatment of falciparum malaria until resistance emerged. CQ is suggested to accumulate in the parasite's digestive vacuole (DV), where it unfolds its anti-malarial properties. Discrepancies of CQ accumulation in CQ-sensitive (CQS) and CQ-resistant (CQR) strains are thought to play a significant role in drug susceptibility. Analysis of CQ transport and intracellular localization using a fluorescently tagged CQ analogue could provide much needed information to distinguish susceptible from resistant parasite strains. The fluorescently tagged CQ analogue LynxTag-CQ™GREEN (CQGREEN) is commercially available and was assessed for its suitability. METHODS: IC50 values were determined for both CQ and CQGREEN in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQGREEN in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQGREEN for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQGREEN uptake rates were determined for the cytosol and DV in the presence and absence of verapamil. RESULTS: In CQS strains, twofold higher IC50 values were determined for the CQGREEN analogue compared to CQ. No significant differences in IC50 values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQGREEN. Live cell imaging revealed that CQGREEN fluorescence was mainly seen in the cytosol of most parasites, independent of the concentration used. Incubation periods of up to 7 h did not influence intracellular localization of CQGREEN. Nevertheless, CQGREEN uptake rates in CQR strains were reduced by 50% compared to CQS strains. CONCLUSION: Although fluorescence of CQGREEN was mainly seen in the cytosol of parasites, IC50 assays showed comparable efficacy of CQGREEN and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQGREEN in CQR strains compared to CQS strains indicate parasite-specific responses to CQGREEN exposure. The data contains valuable information when CQGREEN is used as an analogue for CQ.


Assuntos
Antimaláricos/metabolismo , Cloroquina/metabolismo , Resistência a Medicamentos , Corantes Fluorescentes/metabolismo , Plasmodium falciparum/metabolismo , Transporte Biológico , Cloroquina/análogos & derivados , Plasmodium falciparum/efeitos dos fármacos
4.
PLoS Biol ; 17(10): e3000490, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613878

RESUMO

Many important infectious diseases are the result of zoonoses, in which pathogens that normally infect animals acquire mutations that enable the breaching of species barriers to permit the infection of humans. Our understanding of the molecular events that enable host switching are often limited, and yet this is a fundamentally important question. Plasmodium falciparum, the etiological agent of severe human malaria, evolved following a zoonotic transfer of parasites from gorillas. One gene-rh5-which encodes an essential ligand for the invasion of host erythrocytes, is suspected to have played a critical role in this host switch. Genome comparisons revealed an introgressed sequence in the ancestor of P. falciparum containing rh5, which likely allowed the ancestral parasites to infect both gorilla and human erythrocytes. To test this hypothesis, we resurrected the ancestral introgressed reticulocyte-binding protein homologue 5 (RH5) sequence and used quantitative protein interaction assays to demonstrate that this ancestral protein could bind the basigin receptor from both humans and gorillas. We also showed that this promiscuous receptor binding phenotype of RH5 was shared with the parasite clade that transferred its genome segment to the ancestor of P. falciparum, while the other lineages exhibit host-specific receptor binding, confirming the central importance of this introgression event for Plasmodium host switching. Finally, since its transfer to humans, P. falciparum, and also the RH5 ligand, have evolved a strong human specificity. We show that this subsequent restriction to humans can be attributed to a single amino acid mutation in the RH5 sequence. Our findings reveal a molecular pathway for the origin and evolution of human P. falciparum malaria and may inform molecular surveillance to predict future zoonoses.


Assuntos
Basigina/genética , Proteínas de Transporte/genética , Genoma de Protozoário , Malária Falciparum/transmissão , Malária Falciparum/veterinária , Plasmodium falciparum/genética , Substituição de Aminoácidos , Animais , Basigina/química , Basigina/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Eritrócitos/parasitologia , Expressão Gênica , Gorilla gorilla/parasitologia , História Antiga , Especificidade de Hospedeiro , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/história , Modelos Moleculares , Mutação , Filogenia , Plasmodium falciparum/classificação , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Ligação Proteica , Estrutura Secundária de Proteína , Zoonoses
5.
PLoS Biol ; 17(9): e3000473, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568532

RESUMO

Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites.


Assuntos
Antígenos de Protozoários/metabolismo , Plasmodium falciparum/metabolismo , Aminoácidos/metabolismo , Eritrócitos/parasitologia , Técnicas de Inativação de Genes , Glutationa Transferase/metabolismo , Interações Hospedeiro-Parasita , Nutrientes/metabolismo , Plasmodium falciparum/genética , Vacúolos/metabolismo
6.
Malar J ; 18(1): 349, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619243

RESUMO

BACKGROUND: The resistance of Plasmodium falciparum to artemisinin has been identified in Asia and some parts of Africa. The drug resistance of P. falciparum will be an obstacle to the successful elimination of malaria by 2025. Whole-genome sequencing of the artemisinin-resistant parasite line revealed mutations on the k13 gene associated with drug resistance in P. falciparum. To understand the artemisinin resistance of the imported P. falciparum cases from Africa, the mutations in the k13 gene in parasites from imported malaria cases in Guangxi Province were detected and the treatment efficiency of artesunate monotherapy was observed. METHODS: DNA was extracted from 319 blood samples from migrant workers with P. falciparum infection who returned to their hometown in Guangxi Province from Africa between 2014 and 2017. The k13-propeller gene was amplified by nested PCR, and sequencing, gene mutation frequency and geographic difference of imported P. falciparum cases were analysed by comparison with the wild-type strain. Of 319 patients, 158 were P. falciparum-infected and were treated with intravenous injection of artesunate and were observed, including the time of asexual stage clearance and the dose of artesunate used. RESULTS: Of the 319 P. falciparum samples, 12 samples had the k13-propeller mutation, and 11 point mutations were detected; 5 were non-synonymous mutations (T474I, A481T, A578S, V603E, G665S) and were not associated with artemisinin resistance. The clinical treatment observation showed that the median (IQR) dose of artesunate for peripheral blood parasite asexual stage clearance was 407.55 (360-510) mg, and the D3 parasite clearance rate was 70.25%, including the five k13-propeller mutations of P. falciparum. After 7 days of treatment, 98.73% of cases were cleared. Two cases were treated with artemisinin for 8 days with a 960-mg dose to completely clear the asexual parasite, but they did not have a mutation in the k13 gene. CONCLUSIONS: Five mutations of the k13-propeller gene in 319 P. falciparum samples from patients returning from Africa were identified. The frequency of the k13-propeller mutants was low, and the mutations were not strongly associated with artemisinin resistance. The median (IQR) dose of artesunate monotherapy in actual clinical treatment to remove asexual parasite stages was 407.55 (360-510) mg, equivalent to D3-D4. Some P. falciparum cases without a k13-propeller mutation showed obvious delayed clearance of the parasite from peripheral blood. Trial registration The diagnosis of malaria and the treatment of malaria-infected patients are the routine work of Centres for Disease Control and Prevention. Information on the patients was conveyed with the patient's approval, and the research aim, methods, risks and benefits of the study were explained in detail to the patients.


Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Artesunato/uso terapêutico , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , China , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Migrantes/estatística & dados numéricos
7.
PLoS Pathog ; 15(10): e1008086, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658303

RESUMO

Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design.


Assuntos
Proteína NEDD8/metabolismo , Plasmodium falciparum/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Antimaláricos/farmacologia , Linhagem Celular , Células HEK293 , Humanos , Hidrólise , Malária Falciparum/tratamento farmacológico , Malária Falciparum/patologia , Ubiquitinação/fisiologia
8.
Malar J ; 18(1): 317, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533747

RESUMO

BACKGROUND: Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity of PfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of the PfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among Bioko P. falciparum isolates were analysed. METHODS: 214 blood samples were collected from Bioko Island patients with P. falciparum malaria between 2011 and 2017. A fragment spanning DI of PfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 global PfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program. RESULTS: 131 different haplotypes of PfAMA-1 were identified in 214 Bioko Island P. falciparum isolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified in PfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of Bioko PfAMA-1 DIs were similar to those in global PfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates. CONCLUSIONS: Patterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of global PfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure of PfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.


Assuntos
Antígenos de Protozoários/genética , Variação Genética , Proteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Seleção Genética , Antígenos de Protozoários/metabolismo , Guiné Equatorial , Proteínas de Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
9.
Nat Commun ; 10(1): 4041, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492901

RESUMO

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma, have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium. Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.


Assuntos
Proteínas de Transporte/metabolismo , Organelas/metabolismo , Fosfolipídeos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Exocitose , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Parasitos/metabolismo , Parasitos/ultraestrutura , Fosfolipídeos/química , Proteínas de Protozoários/genética
10.
PLoS Pathog ; 15(9): e1008049, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31491036

RESUMO

The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite.


Assuntos
Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Proteínas de Protozoários/metabolismo , Antígenos de Protozoários/imunologia , Humanos , Malária/metabolismo , Malária Falciparum/metabolismo , Proteínas de Membrana/metabolismo , Merozoítos/metabolismo , Organelas/metabolismo , Plasmodium falciparum/metabolismo , Transporte Proteico/fisiologia
12.
Parasitol Res ; 118(10): 2753-2766, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418110

RESUMO

Plasmodium falciparum (Pf) refurbishes the infected erythrocytes by exporting a myriad of parasite proteins to the host cell. A novel exported protein family 'Plasmodium Helical Interspersed Subtelomeric' (PHIST) has gained attention for its significant roles in parasite biology. Here, we have collected and analysed available information on PHIST members to enhance understanding of their functions, varied localization and structure-function correlation. Functional diversity of PHIST proteins is highlighted by their involvement in PfEMP1 (Pf erythrocyte membrane protein 1) expression, trafficking and switching. This family also contributes to cytoadherence, gametocytogenesis, host cell modification and generation of extracellular vesicles. While the PHIST domain forms the hallmark of this family, existence and functions of additional domains (LyMP, TIGR01639) and the MEC motif underscores its diversity further. Since specific PHIST proteins seem to form pairs with PfEMP1 members, we have used in silico tools to predict such potential partners in Pf. This information and our analysis of structural data on a PHIST member provide important insights into their functioning. This review overall enables readers to view the PHIST family comprehensively, while highlighting key knowledge gaps in the field.


Assuntos
Malária Falciparum/parasitologia , Família Multigênica , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Eritrócitos/parasitologia , Humanos , Plasmodium falciparum/química , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
13.
Nat Commun ; 10(1): 3806, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444345

RESUMO

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.


Assuntos
Engenharia Genética/métodos , Interações Hospedeiro-Parasita , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Reticulócitos/parasitologia , Animais , Basigina/genética , Basigina/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Eritroblastos/fisiologia , Técnicas de Inativação de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Plasmodium falciparum/metabolismo , Domínios Proteicos/genética , Proteínas de Protozoários/metabolismo , Reticulócitos/fisiologia , Transdução Genética
14.
Nanoscale ; 11(32): 15307-15311, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31386727

RESUMO

Vaccines for many important diseases remain elusive, and those for others need to be updated frequently. Vaccine efficacy has been hindered by existing sequence diversity in proteins and by newly-acquired mutations that enable escape from vaccine-induced immune responses. To address these limitations, we developed an approach for nanopatterning protein antigens that combines the site-specific incorporation of non-canonical amino acids with chemical modification to focus the immune response on conserved protein regions. We demonstrated the approach using green fluorescent protein (GFP) as a model antigen and with a promising malaria vaccine candidate, Merozoite surface protein 119 (MSP119). Immunization of mice with nanopatterned MSP119 elicited antibodies that recognized MSP119 from heterologous strains, differing in sequence at as many as 21 of 96 residues. Nanopatterning should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.


Assuntos
Antígenos/imunologia , Nanoestruturas/química , Animais , Anticorpos Antiprotozoários/imunologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/imunologia , Vacinas Antimaláricas/imunologia , Camundongos , Mutagênese , Plasmodium falciparum/metabolismo , Polietilenoglicóis/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
15.
Gene ; 716: 144016, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377318

RESUMO

Drug resistance of malaria parasites remains a problem affecting antimalarial treatment and control of the disease. We previously synthesized an antimalarial endoperoxide, N-89, having high antimalarial effects in vitro and in vivo. In this study we seek to understand the resistant mechanism against N-89 by establishing a highly N-89-resistant clone, named NRC10H, of the Plasmodium falciparum FCR-3 strain. We describe gene mutations in the parent FCR-3 strain and the NRC10H clone using whole-genome sequencing and subsequently by expression profiling using quantitative real-time PCR. Seven genes related to drug resistance, proteolysis, glycophosphatidylinositol anchor biosynthesis, and phosphatidylethanolamine biosynthesis exhibited a single amino acid substitution in the NRC10H clone. Among these seven genes, the multidrug resistance protein 2 (mdr2) variant A532S was found only in NRC10H. The genetic status of the P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC), a potential target of N-89, was similar between the NRC10H clone and the parent FCR-3 strain. These findings suggest that the genetic alterations of the identified seven genes, in particular mdr2, in NRC10H could give rise to resistance of the antimalarial endoperoxide N-89.


Assuntos
Antimaláricos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Compostos de Espiro/farmacologia , Resistência a Medicamentos/genética , Genômica , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Sequenciamento Completo do Genoma
16.
Nat Microbiol ; 4(11): 1990-2000, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31384003

RESUMO

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.


Assuntos
Plasmodium falciparum/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Artemisininas/farmacologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Multimerização Proteica , Proteostase , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
17.
Elife ; 82019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31322501

RESUMO

In addition to its role in erythrocyte invasion, Plasmodium falciparum actin is implicated in endocytosis, cytokinesis and inheritance of the chloroplast-like organelle called the apicoplast. Previously, the inability to visualise filamentous actin (F-actin) dynamics had restricted the characterisation of both F-actin and actin regulatory proteins, a limitation we recently overcame for Toxoplasma (Periz et al, 2017). Here, we have expressed and validated actin-binding chromobodies as F-actin-sensors in Plasmodium falciparum and characterised in-vivo actin dynamics. F-actin could be chemically modulated, and genetically disrupted upon conditionally deleting actin-1. In a comparative approach, we demonstrate that Formin-2, a predicted nucleator of F-actin, is responsible for apicoplast inheritance in both Plasmodium and Toxoplasma, and additionally mediates efficient cytokinesis in Plasmodium. Finally, time-averaged local intensity measurements of F-actin in Toxoplasma conditional mutants revealed molecular determinants of spatiotemporally regulated F-actin flow. Together, our data indicate that Formin-2 is the primary F-actin nucleator during apicomplexan intracellular growth, mediating multiple essential functions.


Assuntos
Citoesqueleto de Actina/metabolismo , Citocinese/genética , Malária Falciparum/genética , Citoesqueleto de Actina/química , Actinas/genética , Actinas/metabolismo , Apicoplastos/química , Apicoplastos/metabolismo , Endocitose/genética , Eritrócitos/química , Eritrócitos/parasitologia , Regulação da Expressão Gênica/genética , Humanos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Ligação Proteica , Toxoplasma/metabolismo , Toxoplasma/patogenicidade
18.
PLoS Biol ; 17(7): e3000376, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31318858

RESUMO

Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the 'delayed death' effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.


Assuntos
Apicoplastos/metabolismo , Espaço Intracelular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/farmacologia , Morte Celular/efeitos dos fármacos , Hemiterpenos/metabolismo , Hemiterpenos/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/parasitologia , Malária Falciparum/parasitologia , Metabolômica/métodos , Compostos Organofosforados/metabolismo , Compostos Organofosforados/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Prenilação de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/parasitologia
19.
Int J Biol Macromol ; 138: 996-1005, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31356937

RESUMO

Several exported Plasmodium falciparum (Pf) proteins contribute to malaria biology through their involvement in cytoadherence, immune evasion and host cell remodelling. Many of these exported proteins and other host molecules are present in iRBC (infected red blood cell) generated extracellular vesicles (EVs), which are responsible for host cell modification and parasite development. CX3CL1 binding proteins (CBPs) present on the surface of iRBCs have been reported to contribute to cytoadhesion by binding with the chemokine 'CX3CL1' via their extracellular domains. Here, we have characterized the cytoplasmic domain of CBP2 to understand its function in parasite biology using biochemical and biophysical methods. Recombinant cytoplasmic CBP2 (cCBP2) binds nucleic acids showing interaction with DNA/RNA. cCBP2 shows dimer formation under non-reducing conditions highlighting the role of disulphide bonds in its oligomerization while ATP binding leads to structural changes in the protein. In vitro interaction studies depict its binding with a Maurer's cleft resident protein 'PfSBP1', which is influenced by ATP binding of cCBP2. Our results suggest CBP2 as a two-transmembrane (2TM) receptor responsible for targeting EVs and delivering cargo to host endothelial cells. We propose CBP2 as an important molecule having roles in cytoadherence and immune modulation through its extracellular and cytoplasmic domains respectively.


Assuntos
Quimiocina CX3CL1/metabolismo , Ácidos Nucleicos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Quimiocina CX3CL1/química , Humanos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química
20.
Chem Commun (Camb) ; 55(64): 9535-9538, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31334508

RESUMO

Endoperoxide-containing antimalarials, such as artemisinin and the synthetic trioxolane OZ439, are prodrugs activated by heme to generate primary and secondary carbon-centered radicals. We employed activity-based protein profiling (ABPP) to show that the secondary-carbon-centered radical of 1,2,4-trioxolanes is primarily responsible for protein labeling in malaria parasites.


Assuntos
Carbono/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais
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