Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.481
Filtrar
1.
Front Cell Infect Microbiol ; 11: 669088, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34268135

RESUMO

The human malaria parasite Plasmodium falciparum expresses variant PfEMP1 proteins on the infected erythrocyte, which function as ligands for endothelial receptors in capillary vessels, leading to erythrocyte sequestration and severe malaria. The factors that orchestrate the mono-allelic expression of the 45-90 PfEMP1-encoding var genes within each parasite genome are still not fully identified. Here, we show that the transcription factor PfAP2-O influences the transcription of var genes. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence in CD36 adherent parasites. AP2-O-knocked-down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is, beside its role in transmission stages, also one of the virulence gene transcriptional regulators and may therefore be exploited as an important target to disrupt severe malaria and block parasite transmission.


Assuntos
Malária Falciparum , Plasmodium falciparum , Animais , Eritrócitos , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Desenvolvimento Sexual , Fatores de Transcrição/genética , Transcrição Genética , Virulência/genética
2.
Front Cell Infect Microbiol ; 11: 654216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34262880

RESUMO

Malaria transmission relies on parasite-mosquito midgut interaction. The interactive proteins are hypothesized to be ideal targets to block malaria transmission to mosquitoes. We chose 76 genes that contain signal peptide-coding regions and are upregulated and highly abundant at sexual stages. Forty-six of these candidate genes (60%) were cloned and expressed using the baculovirus expression system in insect cells. Six of them, e.g., PF3D7_0303900, PF3D7_0406200 (Pfs16), PF3D7_1204400 (Pfs37), PF3D7_1214800, PF3D7_1239400, and PF3D7_1472800 were discovered to interact with blood-fed mosquito midgut lysate. Previous works showed that among these interactive proteins, knockout the orthologs of Pfs37 or Pfs16 in P. berghei reduced oocysts in mosquitoes. Here we further found that anti-Pfs16 polyclonal antibody significantly inhibited P. falciparum transmission to Anopheles gambiae. Investigating these candidate proteins will improve our understanding of malaria transmission and discover new targets to break malaria transmission.


Assuntos
Malária , Parasitos , Plasmodium , Animais , Mosquitos Vetores , Plasmodium falciparum/genética
3.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068393

RESUMO

The highly complex life cycle of the human malaria parasite, Plasmodium falciparum, is based on an orchestrated and tightly regulated gene expression program. In general, eukaryotic transcription regulation is determined by a combination of sequence-specific transcription factors binding to regulatory DNA elements and the packaging of DNA into chromatin as an additional layer. The accessibility of regulatory DNA elements is controlled by the nucleosome occupancy and changes of their positions by an active process called nucleosome remodeling. These epigenetic mechanisms are poorly explored in P. falciparum. The parasite genome is characterized by an extraordinarily high AT-content and the distinct architecture of functional elements, and chromatin-related proteins also exhibit high sequence divergence compared to other eukaryotes. Together with the distinct biochemical properties of nucleosomes, these features suggest substantial differences in chromatin-dependent regulation. Here, we highlight the peculiarities of epigenetic mechanisms in P. falciparum, addressing chromatin structure and dynamics with respect to their impact on transcriptional control. We focus on the specialized chromatin remodeling enzymes and discuss their essential function in P. falciparum gene regulation.


Assuntos
Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação da Expressão Gênica , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Transcrição Genética , Animais , Humanos , Estágios do Ciclo de Vida
4.
J Pharm Biomed Anal ; 203: 114178, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34082143

RESUMO

Malaria infection represents a major public health and economic issue that leads to morbidity and mortality globally. A highly effective and uncomplicated detection tool is required for malaria control in geographical hotspots of transmission. We developed a simple and more sensitive novel approach for the detection of the 18S rRNA gene of Plasmodium falciparum based on loop-mediated isothermal amplification (LAMP) and visualization using colorimetric, streptavidin-functionalized gold nanoparticles (SA-GNPs). Two loop primers of LAMP were biotinylated to produce biotin-containing products during amplification. After the addition of SA-GNPs, clusters of avidin-biotin complexes were established in the LAMP structure. While the positive reactions remained wine red, the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C). All steps of the assay were completed within 50 min, its detection limit was 1 parasite/µL, and it was highly specific for P. falciparum. This effortless detection system with high sensitivity and specificity could provide an alternative choice for malaria diagnostics in resource-limited regions.


Assuntos
Nanopartículas Metálicas , Plasmodium falciparum , Ouro , Temperatura Alta , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Plasmodium falciparum/genética , Sensibilidade e Especificidade
5.
Front Cell Infect Microbiol ; 11: 673966, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34079769

RESUMO

It is widely accepted that the structure of RNA plays important roles in a number of biological processes, such as polyadenylation, splicing, and catalytic functions. Dynamic changes in RNA structure are able to regulate the gene expression programme and can be used as a highly specific and subtle mechanism for governing cellular processes. However, the nature of most RNA secondary structures in Plasmodium falciparum has not been determined. To investigate the genome-wide RNA secondary structural features at single-nucleotide resolution in P. falciparum, we applied a novel high-throughput method utilizing the chemical modification of RNA structures to characterize these structures. Structural data from parasites are in close agreement with the known 18S ribosomal RNA secondary structures of P. falciparum and can help to predict the in vivo RNA secondary structure of a total of 3,396 transcripts in the ring-stage and trophozoite-stage developmental cycles. By parallel analysis of RNA structures in vivo and in vitro during the Plasmodium parasite ring-stage and trophozoite-stage intraerythrocytic developmental cycles, we identified some key regulatory features. Recent studies have established that the RNA structure is a ubiquitous and fundamental regulator of gene expression. Our study indicate that there is a critical connection between RNA secondary structure and mRNA abundance during the complex biological programme of P. falciparum. This work presents a useful framework and important results, which may facilitate further research investigating the interactions between RNA secondary structure and the complex biological programme in P. falciparum. The RNA secondary structure characterized in this study has potential applications and important implications regarding the identification of RNA structural elements, which are important for parasite infection and elucidating host-parasite interactions and parasites in the environment.


Assuntos
Malária Falciparum , Plasmodium falciparum , Humanos , Plasmodium falciparum/genética , Poliadenilação , Proteínas de Protozoários/genética , RNA/genética , RNA Mensageiro/metabolismo
6.
Front Public Health ; 9: 649170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937177

RESUMO

Increased population movement has increased the risk of reintroducing parasites to elimination areas and also dispersing drug-resistant parasites to new regions. Therefore, reliable and repeatable methods to trace back to the source of imported infections are essential. The recently developed 23-single-nucleotide polymorphism (SNP) barcode from organellar genomes of mitochondrion (mt) and apicoplast (apico) provides a valuable tool to locate the geographic origin of Plasmodium falciparum. This study aims to explore the feasibility of using the 23-SNP barcode for tracking P. falciparum by polymerase chain reaction and sequencing, while providing geographical haplotypes of isolates that originated from Central Africa. Based on 23-SNP barcode analysis, SNPs were found at seven loci; 27 isolates were confirmed to have originated in West Africa, and this study also showed four isolates from Central Africa (Equatorial Guinea, 3; Republic of Congo, 1) that originated in East Africa. This study provides the sequence data from Central Africa and fills 23-SNP barcode data gaps of sample origins.


Assuntos
Plasmodium falciparum , África Oriental , África Ocidental , Congo , Guiné Equatorial , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase
7.
Nat Commun ; 12(1): 2956, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011972

RESUMO

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.


Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Malária Falciparum/complicações , Placenta/parasitologia , Complicações Parasitárias na Gravidez/metabolismo , Complicações Parasitárias na Gravidez/parasitologia , Sequência de Aminoácidos , Antígenos de Protozoários/genética , Microscopia Crioeletrônica , Feminino , Humanos , Evasão da Resposta Imune , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Placenta/imunologia , Placenta/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Gravidez , Ligação Proteica , Domínios Proteicos
8.
BMC Infect Dis ; 21(1): 439, 2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-33985447

RESUMO

BACKGROUND: Genetic diversity in Plasmodium falciparum populations can be used to describe the resilience and spatial distribution of the parasite in the midst of intensified intervention efforts. This study used microsatellite analysis to evaluate the genetic diversity and population dynamics of P. falciparum parasites circulating in three ecological zones of Ghana. METHODS: A total of 1168 afebrile children aged between 3 to 13 years were recruited from five (5) Primary schools in 3 different ecological zones (Sahel (Tamale and Kumbungu), Forest (Konongo) and Coastal (Ada and Dodowa)) of Ghana. Asymptomatic malaria parasite carriage was determined using microscopy and PCR, whilst fragment analysis of 6 microsatellite loci was used to determine the diversity and population structure of P. falciparum parasites. RESULTS: Out of the 1168 samples examined, 16.1 and 39.5% tested positive for P. falciparum by microscopy and nested PCR respectively. The genetic diversity of parasites in the 3 ecological zones was generally high, with an average heterozygosity (He) of 0.804, 0.787 and 0.608 the rainy (peak) season for the Sahel, Forest and Coastal zones respectively. The mean He for the dry (off-peak) season were 0.562, 0.693 and 0.610 for the Sahel, Forest and Coastal zones respectively. Parasites from the Forest zone were more closely related to those from the Sahel than from the Coastal zone, despite the Coastal zone being closer in physical distance to the Forest zone. The fixation indexes among study sites ranged from 0.049 to 0.112 during the rainy season and 0.112 to 0.348 during the dry season. CONCLUSION: A large asymptomatic parasite reservoir was found in the school children during both rainy and dry seasons, especially those in the Forest and Sahel savannah zones where parasites were also found to be related compared to those from the Coastal zone. Further studies are recommended to understand why despite the roll out of several malaria interventions in Ghana, high transmission still persist.


Assuntos
Portador Sadio/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Adolescente , Portador Sadio/epidemiologia , Criança , Pré-Escolar , DNA de Protozoário/genética , Feminino , Variação Genética , Genética Populacional , Gana/epidemiologia , Humanos , Malária Falciparum/epidemiologia , Masculino , Repetições de Microssatélites/genética , Plasmodium falciparum/citologia , Plasmodium falciparum/isolamento & purificação , Estações do Ano
9.
Int J Infect Dis ; 107: 234-241, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33940188

RESUMO

BACKGROUND: Recent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region. METHOD: In this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13, crt, mdr1, dhfr, and dhps, and the cytochrome b gene (cytb) in Plasmodium falciparum. RESULTS: No confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other nonsynonymous mutations was observed across all the genes, except for Pfcytb, suggesting continued selection pressure. CONCLUSIONS: The results from this study supported the continued use of artemisinin-based combination therapy and administration of sulfadoxine-pyrimethamine for intermittent preventive therapy in pregnant women, and for seasonal chemoprevention in these study sites in Cameroon.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Marcadores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Alelos , Camarões , Feminino , Genótipo , Humanos , Mutação , Plasmodium falciparum/isolamento & purificação , Gravidez
10.
Nat Commun ; 12(1): 3196, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045457

RESUMO

Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonization of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and non-conserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, provide a fine-grained atlas that enables intensive investigation of the P. falciparum transcriptional journey. As well as providing insights into gene function across the transmission cycle, the atlas opens the door for identification of drug and vaccine targets to stop malaria transmission and thereby prevent disease.


Assuntos
Anopheles/parasitologia , Estágios do Ciclo de Vida/genética , Malária Falciparum/transmissão , Mosquitos Vetores/parasitologia , Plasmodium falciparum/genética , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Feminino , Interações Hospedeiro-Parasita/genética , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Masculino , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , RNA-Seq , Análise de Célula Única , Especificidade da Espécie , Transcriptoma/efeitos dos fármacos
11.
Trans R Soc Trop Med Hyg ; 115(5): 531-537, 2021 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-33823558

RESUMO

BACKGROUND: In Nigeria, indiscriminate use of antimalarial drugs may contribute to the threat of drug resistance, but this has not been evaluated among people living with human immunodeficiency virus (HIV). METHODS: HIV-positive adults attending a university hospital HIV clinic and HIV-negative adult volunteers from the university hospital community with a positive blood film were treated with artemether-lumefantrine. Parasite DNA from before and after treatment was polymerase chain reaction amplified to identify molecular markers of drug susceptibility. RESULTS: The pfcrt76T genotype was prevalent among both HIV-positive and HIV-negative participants (78.6% and 68.2%, respectively). Three new mutations in the pfmdr1 gene-F73S, S97L and G165R-and the uncommon pfdhps S436F variant were detected, whereas pfdhps K540E and pfdhfr I164L were absent. The A437G allele of pfdhps predominated (62/66 [94%]). The I431 V mutation was found in 19 of 66 pretreatment pfdhps sequences (28.8%). The pfmdr1 86N allele was significantly more common at day 3 post-treatment than at baseline (odds ratio 8.77 [95% confidence interval 1.21 to 380]). CONCLUSIONS: We found evidence of continued chloroquine use among HIV-positive individuals. Selection for the pfmdr1 86N after artemether-lumefantrine treatment was observed, indicating a possible threat to antimalarial efficacy in the study area. The complexity of pfdhps haplotypes emphasises the need for careful monitoring of anti-folate susceptibility in Nigeria.


Assuntos
Antimaláricos , Malária Falciparum , Malária , Adulto , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemeter/uso terapêutico , Combinação Arteméter e Lumefantrina/uso terapêutico , Combinação de Medicamentos , Resistência a Medicamentos/genética , HIV , Humanos , Malária/complicações , Malária/tratamento farmacológico , Malária Falciparum/complicações , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Nigéria , Plasmodium falciparum/genética , Proteínas de Protozoários/genética
12.
Antimicrob Agents Chemother ; 65(7): e0027521, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33875422

RESUMO

Malaria remains one of the deadliest diseases in Africa, particularly for children. While successful in reducing morbidity and mortality, antimalarial treatments are also a major cause of adverse drug reactions (ADRs). Host genetic variation in genes involved in drug disposition or toxicity constitutes an important determinant of ADR risk and can prime for parasite drug resistance. Importantly, however, the genetic diversity in Africa is substantial, and thus, genetic profiles in one population cannot be reliably extrapolated to other ethnogeographic groups. Gabon is considered a high-transmission country, with more than 460,000 malaria cases per year. Yet the pharmacogenetic landscape of the Gabonese population or its neighboring countries has not been analyzed. Using targeted sequencing, here, we profiled 21 pharmacogenes with importance for antimalarial treatment in 48 Gabonese pediatric patients with severe Plasmodium falciparum malaria. Overall, we identified 347 genetic variants, of which 18 were novel, and each individual was found to carry 87.3 ± 9.2 (standard deviation [SD]) variants across all analyzed genes. Importantly, 16.7% of these variants were population specific, highlighting the need for high-resolution pharmacogenomic profiling. Between one in three and one in six individuals harbored reduced-activity alleles of CYP2A6, CYP2B6, CYP2D6, and CYP2C8 with important implications for artemisinin, chloroquine, and amodiaquine therapy. Furthermore, one in three patients harbored at least one G6PD-deficient allele, suggesting a considerably increased risk of hemolytic anemia upon exposure to aminoquinolines. Combined, our results reveal the unique genetic landscape of the Gabonese population and pinpoint the genetic basis for interindividual differences in antimalarial drug responses and toxicity.


Assuntos
Antimaláricos , Malária Falciparum , Malária , Antimaláricos/efeitos adversos , Criança , Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Gabão , Humanos , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética
13.
Antimicrob Agents Chemother ; 65(7): e0271720, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33903109

RESUMO

The artemisinin-based combination therapies (ACTs) used to treat Plasmodium falciparum in Africa are threatened by the emergence of parasites in Asia that carry variants of the Kelch 13 (K13) locus with delayed clearance in response to ACTs. Single nucleotide polymorphisms (SNPs) in other molecular markers, such as ap2mu and ubp1, were associated with artemisinin resistance in rodent malaria and clinical failure in African malaria patients. Here, we characterized the polymorphisms in pfmdr1, pfcrt, pfK13, pfubp1, and pfap2mu among African isolates reported in Shandong and Guangxi provinces in China. Among 144 patients with P. falciparum returning from Africa from 2014 to 2018, pfmdr1 N86Y (8.3%) and pfcrt K76T (2.1%) were the major mutant alleles. The most common genotype for pfcrt was I74E75T76 (8.3%), followed by E75T76 (2.1%). For K13 polymorphisms, a limited number of mutated alleles were observed, and A578S was the most frequently detected allele in 3 isolates (2.1%). A total of 27.1% (20/144) of the isolates were found to contain pfubp1 mutations, including 6 nonsynonymous and 2 synonymous mutations. The pfubp1 genotypes associated with artemisinin resistance were D1525E (10.4%) and E1528D (8.3%). Furthermore, 11 SNPs were identified in pfap2mu, and S160N was the major polymorphism (4.2%). Additionally, 4 different types of insertions were found in pfap2mu, and the codon AAT, encoding aspartic acid, was more frequently observed at codons 226 (18.8%) and 326 (10.7%). Moreover, 4 different types of insertions were observed in pfubp1 at codon 1520, which was the most common (6.3%). These findings indicate a certain degree of variation in other potential molecular markers, such as pfubp1 and pfap2mu, and their roles in either the parasite's mechanism of resistance or the mode of action should be evaluated or elucidated further.


Assuntos
Antimaláricos , Malária Falciparum , África , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Ásia , China , Resistência a Medicamentos/genética , Humanos , Malária Falciparum/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
14.
Molecules ; 26(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33921170

RESUMO

Malaria is one of the most life-threatening infectious diseases and constitutes a major health problem, especially in Africa. Although artemisinin combination therapies remain efficacious to treat malaria, the emergence of resistant parasites emphasizes the urgent need of new alternative chemotherapies. One strategy is the repurposing of existing drugs. Herein, we reviewed the antimalarial effects of marketed antibiotics, and described in detail the fast-acting antibiotics that showed activity in nanomolar concentrations. Antibiotics have been used for prophylaxis and treatment of malaria for many years and are of particular interest because they might exert a different mode of action than current antimalarials, and can be used simultaneously to treat concomitant bacterial infections.


Assuntos
Antimaláricos/uso terapêutico , Reposicionamento de Medicamentos/métodos , Animais , Antibacterianos/uso terapêutico , Resistência a Medicamentos/genética , Humanos , Malária/fisiopatologia , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade
15.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670016

RESUMO

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins' function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.


Assuntos
Atovaquona/farmacologia , Citocromos b/genética , Resistência a Medicamentos/genética , Proteínas Ferro-Enxofre/genética , Bicamadas Lipídicas/metabolismo , Mutação/genética , Fosfolipídeos/metabolismo , Plasmodium falciparum/genética , Animais , Atovaquona/química , Domínio Catalítico , Bovinos , Resistência a Medicamentos/efeitos dos fármacos , Entropia , Proteínas Ferro-Enxofre/metabolismo , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Plasmodium falciparum/efeitos dos fármacos , Conformação Proteica , Mapas de Interação de Proteínas , Estabilidade Proteica
16.
Malar J ; 20(1): 124, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653360

RESUMO

BACKGROUND: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. METHODS: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. RESULTS: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure. CONCLUSIONS: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.


Assuntos
Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Epitopos , Guiné Equatorial/epidemiologia , Frequência do Gene , Variação Genética , Haplótipos , Humanos , Vacinas Antimaláricas , Malária Falciparum/epidemiologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Polimorfismo Genético , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Seleção Genética
17.
EMBO Rep ; 22(5): e51660, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33665945

RESUMO

Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender-specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2-O3 operates as a transcription repressor in the female gametocytes. AP2-O3 is specifically expressed in the female gametocytes. AP2-O3-deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post-fertilization. AP2-O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2-O3 targets a substantial proportion of the male genes by recognizing an 8-base DNA motif. In addition, the maternal AP2-O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female-specific transcriptome and essential for the mosquito transmission of Plasmodium.


Assuntos
Plasmodium berghei , Plasmodium falciparum , Animais , Feminino , Gametogênese/genética , Masculino , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Fatores de Transcrição/genética , Transcriptoma
18.
Artigo em Inglês | MEDLINE | ID: mdl-33722883

RESUMO

Conventional methods utilizing in vitro protein activity assay or in vivo parasite survival to screen for malaria inhibitors suffer from high experimental background and/or inconvenience. Here, we introduce a yeast-based system to facilitate chemical screening for specific protein or pathway inhibitors. The platform comprises several isogeneic Pichia strains that differ only in the target of interest, so that a compound which inhibits one strain but not the other is implicated in working specifically against the target. We used Plasmodium falciparum NDH2 (PfNDH2), a type II NADH dehydrogenase, as a proof of principle to show how well this works. Three isogenic Pichia strains harboring, respectively, exogeneously introduced PfNDH2, its own complex I (a type I NADH dehydrogenase), and PfNDH2 with its own complex I, were constructed. In a pilot screen of more than 2,000 compounds, we identified a highly specific inhibitor that acts on PfNDH2. This compound poorly inhibits the parasites at the asexual blood stage; however, is highly effective in repressing oocyst maturation in the mosquito stage. Our results demonstrate that the yeast cell-based screen platform is feasible, efficient, economical, and has very low background noise. Similar strategies could be extended to the functional screen for interacting molecules of other targets.


Assuntos
Antimaláricos , Malária Falciparum , Malária , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Descoberta de Drogas , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , NADH Desidrogenase/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
ACS Infect Dis ; 7(4): 811-825, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33715347

RESUMO

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 (pfmfr3) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.


Assuntos
Antimaláricos , Malária , Antimaláricos/farmacologia , Resistência a Medicamentos , Humanos , Malária/tratamento farmacológico , Mutação , Plasmodium falciparum/genética
20.
Front Cell Infect Microbiol ; 11: 631545, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33708645

RESUMO

Malaria, an infectious disease caused by Plasmodium parasites, still accounts for amounts of deaths annually in last decades. Despite the significance of Plasmodium falciparum as a model organism of malaria parasites, our understanding of gene expression of this parasite remains largely elusive since lots of progress on its genome and transcriptome are based on assembly with short sequencing reads. Herein, we report the new version of transcriptome dataset containing all full-length transcripts over the whole asexual blood stages by adopting a full-length sequencing approach with optimized experimental conditions of cDNA library preparation. We have identified a total of 393 alternative splicing (AS) events, 3,623 long non-coding RNAs (lncRNAs), 1,555 alternative polyadenylation (APA) events, 57 transcription factors (TF), 1,721 fusion transcripts in P. falciparum. Furthermore, the shotgun proteome was performed to validate the full-length transcriptome of P. falciparum. More importantly, integration of full-length transcriptomic and proteomic data identified 160 novel small proteins in lncRNA regions. Collectively, this full-length transcriptome dataset with high quality and accuracy and the shotgun proteome analyses shed light on the complex gene expression in malaria parasites and provide a valuable resource for related functional and mechanistic researches on P. falciparum genes.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Plasmodium falciparum , Processamento Alternativo , Perfilação da Expressão Gênica , Plasmodium falciparum/genética , Proteômica , Transcriptoma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...