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1.
Artigo em Inglês | MEDLINE | ID: mdl-33533814

RESUMO

Malaria represents a serious public health problem, presenting with high rates of incidence, morbidity and mortality in tropical and subtropical regions of the world. According to the World Health Organization, in 2018 there were 228 million cases and 405 thousand deaths caused by this disease in the world, affecting mainly children and pregnant women in Africa. Despite the programs carried out to control this disease, drug resistance and invertebrate vector resistance to insecticides have generated difficulties. An efficient vaccine against malaria would be a strategy with a high impact on the eradication and control of this disease. Researches aimed at developing vaccines have focused on antigens of high importance for the survival of the parasite such as the Circumsporozoite Surface Protein, involved in the pre-erythrocytic cycle during parasites invasion in hepatocytes. Currently, RTS'S is the most promising vaccine for malaria and was constructed using CSP; its performance was evaluated using two types of adjuvants: AS01 and AS02. The purpose of this review was to provide a bibliographic survey of historical researches that led to the development of RTS'S and its performance analysis over the decade. The search for new adjuvants to be associated with this antigen seems to be a way to obtain higher percentages of protection for a future malaria vaccine.


Assuntos
Vacinas Antimaláricas/uso terapêutico , Malária/prevenção & controle , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários , Humanos , Malária/parasitologia , Vacinas Antimaláricas/administração & dosagem , Proteínas de Membrana
2.
Nat Commun ; 12(1): 120, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402698

RESUMO

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.


Assuntos
Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/química , Subunidades Proteicas/química , Proteínas de Protozoários/química , Toxoplasma/ultraestrutura , Sítios de Ligação , Cardiolipinas/química , Cardiolipinas/metabolismo , Microscopia Crioeletrônica , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Termodinâmica , Toxoplasma/genética , Toxoplasma/metabolismo
3.
Nat Commun ; 12(1): 530, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483501

RESUMO

The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite's intra-erythrocytic developmental program. These changes impact cell-cycle periodicity, the unfolded protein response, protein degradation, vesicular trafficking, and mitochondrial metabolism. K13-mediated artemisinin resistance in the Cambodian Cam3.II line was reversed by atovaquone, a mitochondrial electron transport chain inhibitor. These results suggest that mitochondrial processes including damage sensing and anti-oxidant properties might augment the ability of mutant K13 to protect P. falciparum against artemisinin action by helping these parasites undergo temporary quiescence and accelerated growth recovery post drug elimination.


Assuntos
Artemisininas/farmacologia , Resistência a Medicamentos/genética , Eritrócitos/metabolismo , Mutação , Plasmodium falciparum/genética , Antimaláricos/farmacologia , Atovaquona/farmacologia , Pontos de Checagem do Ciclo Celular/genética , Eritrócitos/parasitologia , Perfilação da Expressão Gênica/métodos , Humanos , Metabolômica/métodos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Genéticos , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Proteômica/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
4.
Nat Commun ; 12(1): 116, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33414462

RESUMO

Apicomplexan parasites have evolved efficient and distinctive strategies for intracellular replication where the timing of emergence of the daughter cells (budding) is a decisive element. However, the molecular mechanisms that provide the proper timing of parasite budding remain unknown. Using Toxoplasma gondii as a model Apicomplexan, we identified a master regulator that controls the timing of the budding process. We show that an ApiAP2 transcription factor, TgAP2IX-5, controls cell cycle events downstream of centrosome duplication. TgAP2IX-5 binds to the promoter of hundreds of genes and controls the activation of the budding-specific cell cycle expression program. TgAP2IX-5 regulates the expression of specific transcription factors that are necessary for the completion of the budding cycle. Moreover, TgAP2IX-5 acts as a limiting factor that ensures that asexual proliferation continues by promoting the inhibition of the differentiation pathway. Therefore, TgAP2IX-5 is a master regulator that controls both cell cycle and developmental pathways.


Assuntos
Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/fisiologia , Proliferação de Células , Centrossomo , Replicação do DNA , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Organismos Geneticamente Modificados , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Exp Parasitol ; 222: 108065, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33428893

RESUMO

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.


Assuntos
Epitopos Imunodominantes/isolamento & purificação , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Proteômica/métodos , Sequência de Aminoácidos , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/isolamento & purificação , Humanos , Immunoblotting , Leishmaniose Visceral/imunologia , Conformação Molecular , Estrutura Secundária de Proteína , Proteômica/normas , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Testes Sorológicos/normas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
J Proteomics ; 234: 104083, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33373718

RESUMO

Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2ß3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment. BIOLOGICAL SIGNIFICANCE: Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems. We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies. This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.


Assuntos
Antígenos CD36 , Enzimas Reparadoras do DNA , Eritrócitos , Molécula 1 de Adesão Intercelular , Venenos de Moluscos , Plasmodium falciparum , Fatores de Transcrição , Animais , Antígenos CD36/química , Antígenos CD36/metabolismo , Caramujo Conus , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Eritrócitos/química , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/metabolismo , Venenos de Moluscos/química , Venenos de Moluscos/farmacologia , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Domínios Proteicos , Proteínas de Protozoários , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
7.
Arch Biochem Biophys ; 698: 108731, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359563

RESUMO

Microbial pathogens, such as Trypanosoma brucei, have an enormous impact on global health and economic systems. Protein kinase A of T. brucei is an attractive drug target as it is an essential enzyme which differs significantly from its human homolog. The hinge region of this protein's regulatory domain is vital for enzymatic function, but its conformation is unknown. Here, the secondary structure of this region has been characterized using NMR and CD spectroscopies. More specifically, three overlapping peptides corresponding to residues T187-I211, G198-Y223 and V220-S245 called peptide 1, peptide 2 and peptide 3, respectively, were studied. The peptide 1 and peptide 2 are chiefly unfolded; only low populations (<10%) of α-helix were detected under the conditions studied. In contrast, the peptide 3 contains a long α-helix whose population is significantly higher; namely, 36% under the conditions studied. Utilizing the dihedral φ and ψ angles calculated on the basis of the NMR data, the conformation of the peptide 3 was calculated and revealed an α-helix spanning residues E230-N241. This α-helix showed amphiphilicity and reversible unfolding and refolding upon heating and cooling. Most fascinating, however, is its capacity to inhibit the activity of the catalytic domain of Trypanosoma equiperdum protein kinase A even though it is quite distinct from the canonical inhibitor motif. Based on this property, we advance that peptoids based on the peptide 3 α-helix could be novel leads for developing anti-trypanosomal therapeutics.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Fragmentos de Peptídeos/química , Inibidores de Proteínas Quinases/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ensaios Enzimáticos , Conformação Proteica em alfa-Hélice , Redobramento de Proteína , Desdobramento de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Alinhamento de Sequência , Suínos
8.
Nucleic Acids Res ; 49(1): 568-583, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332555

RESUMO

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.


Assuntos
Proteínas de Protozoários/química , Estabilidade de RNA/fisiologia , RNA Guia/metabolismo , RNA de Protozoário/metabolismo , Ribonuclease III/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Regulação da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease III/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Dedos de Zinco
9.
Exp Parasitol ; 221: 108060, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33338467

RESUMO

Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.


Assuntos
Ceratite por Acanthamoeba/metabolismo , Acanthamoeba castellanii/metabolismo , Proteínas de Protozoários/biossíntese , Ceratite por Acanthamoeba/parasitologia , Análise de Variância , Animais , Modelos Animais de Doenças , Humanos , Masculino , Proteômica , Proteínas de Protozoários/genética , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Eletroforese em Gel Diferencial Bidimensional
10.
PLoS Genet ; 16(12): e1009268, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33382691

RESUMO

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.


Assuntos
Di-Hidropteroato Sintase/genética , Resistência a Medicamentos , GTP Cicloidrolase/genética , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Pirimetamina/farmacologia , Sulfadoxina/farmacologia
11.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(6): 612-617, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-33325196

RESUMO

OBJECTIVE: To investigate the drug-resistant gene polymorphisms in Plasmodium falciparum imported from Equatorial Guinea to Shandong Province. METHODS: From 2015 to 2016, blood samples were collected from imported P. falciparum malaria patients returning from Equatorial Guinea to Shandong Province, and genome DNA of the malaria parasite was extracted. The drug-resistant Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and K13 genes of P. falciparum were amplified using a PCR assay, followed by DNA sequencing, and the sequences were aligned. RESULTS: The target fragments of all 5 drug-resistant genes of P. falciparum were successfully amplified and sequenced. There were 72.8%, 18.6%, and 8.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfcrt gene, respectively, and all mutant haplotypes were CVIET (the underline indicates the mutation site). There were 20.0%, 61.4% and 18.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfmdr1 gene, respectively, and the mutant haplotypes mainly included YF and NF (the underlines indicate the mutation sites). There were 1.4%, 98.6%, and 0 of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhfr gene, respectively, and AIRNI was the predominant mutant haplotype (the underline indicates the mutation site). There were 1.4%, 94.3%, and 4.3% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhps gene, respectively, and SGKAA was the predominant mutant haplotype (the underline indicates the mutation site). The complete drug-resistant IRNGE genotype consisted of 8.6% of the Pfdhfr and Pfdhps genes, and the K13 gene A578S mutation occurred in 1.4% of the parasite samples. CONCLUSIONS: There are mutations in the Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and K13 genes of P. falciparum imported from Equatorial Guinea to Shandong Province, with a low frequency in the Pfcrt gene mutation and a high frequency in the Pfmdr1, Pfdhfr, and Pfdhps gene mutations, and the K13 gene A578S mutation is detected in the parasite samples.


Assuntos
Antimaláricos , Resistência a Medicamentos/genética , Malária Falciparum , Plasmodium falciparum/genética , Proteínas de Protozoários , Antimaláricos/uso terapêutico , China/epidemiologia , DNA de Protozoário/genética , Guiné Equatorial/epidemiologia , Genótipo , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Mutação , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo Genético/efeitos dos fármacos , Proteínas de Protozoários/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-33375379

RESUMO

Histidine-rich proteins 2 and 3 gene (pfhrp2 and pfhrp3) deletions affect the efficacy of rapid diagnostic tests (RDTs) based on the histidine-rich protein 2 (HRP2), compromising the correct identification of the Plasmodium falciparum species. Therefore, molecular surveillance is necessary for the investigation of the actual prevalence of this phenomenon and the extent of the disappearance of these genes in these areas and other South American countries, thus guiding national malaria control programs on the appropriate use of RDTs. This study aimed to evaluate the pfhrp2 and pfhrp3 gene deletion in P. falciparum in endemic areas of the Brazilian Amazon. Aliquots of DNA from the biorepository of the Laboratory of Basic Research in Malaria, Evandro Chagas Institute, with a positive diagnosis for P. falciparum infection as determined by microscopy and molecular assays, were included. Monoinfection was confirmed by nested-polymerase chain reaction assay, and DNA quality was assessed by amplification of the merozoite surface protein-2 gene (msp2). The pfhrp2 and pfhrp3 genes were amplified using primers for the region between exons 1 and 2 and for all extension of exon 2. Aliquots of DNA from 192 P. falciparum isolates were included in the study, with 68.7% (132/192) from the municipality of Cruzeiro do Sul (Acre) and 31.3% (60/192) from Manaus (Amazonas). Of this total, 82.8% (159/192) of the samples were considered of good quality. In the state of Acre, 71.7% (71/99) showed pfhrp2 gene deletion and 94.9% (94/99) showed pfhrp3 gene deletion, while in the state of Amazonas, 100.0% (60/60) of the samples showed pfhrp2 gene deletion and 98.3% (59/60) showed pfhrp3 gene deletion. Moreover, 79.8% (127/159) of isolates displayed gene deletion. Our findings confirm the presence of a parasite population with high frequencies of pfhrp2 and pfhrp3 gene deletions in the Brazilian Amazon region. This suggests reconsidering the use of HRP2-based RDTs in the Acre and Amazonas states and calls attention to the importance of molecular surveillance and mapping of pfhrp2/pfhrp3 deletions in this area and in other locations in the Amazon region to guarantee appropriate patient care, control and ultimately contribute to achieving P. falciparum malaria elimination.


Assuntos
Plasmodium falciparum/genética , Proteínas/genética , Proteínas de Protozoários , Antígenos de Protozoários/genética , Brasil/epidemiologia , Testes Diagnósticos de Rotina , Deleção de Genes , Humanos , Malária Falciparum , Proteínas de Protozoários/genética
13.
PLoS Genet ; 16(12): e1009266, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370279

RESUMO

Malaria continues to impose a significant health burden in the continent of Africa with 213 million cases in 2018 alone, representing 93% of cases worldwide. Because of high transmission of malaria within the continent, the selection pressures to develop drug resistance in African parasites are distinct compared to the rest of the world. In light of the spread of resistance to artemisinin conferred by the C580Y mutation in the PfKelch13 propeller domain in Southeast Asia, and its independent emergence in South America, it is important to study genetic determinants of resistance in the African context using African parasites. Through in vitro evolution of Senegalese parasites, we had previously generated the artemisinin-resistant parasites Pikine_R and Thiès_R and established pfcoronin mutations to be sufficient to confer artemisinin resistance in the standard ring-stage survival assay (RSA). In the current study, we used genetic analysis of revertants to demonstrate pfcoronin to be the major driver of elevated RSA in the artemisinin-resistant parasites Pikine_R and Thiès_R evolved in vitro. We interrogated the role of a second gene PF3D7_1433800, which also had mutations in both the Pikine_R and Thiès_R selected lines, but found no evidence of a contribution to reduced susceptibility in the RSA survival assay. Nevertheless, our genetic analysis demonstrates that parasite genetic background is important in the level of pfcoronin mediated RSA survival, and therefore we cannot rule out a role for PF3D7_1433800 in other genetic backgrounds. Finally, we tested the potential synergy between the mutations of pfcoronin and pfkelch13 through the generation of single and double mutants in the Pikine genetic background and found that the contribution of pfcoronin to reduced susceptibility is masked by the presence of pfkelch13. This phenomenon was also observed in the 3D7 background, suggesting that pfcoronin may mediate its effects via the same pathway as pfkelch13. Investigating the biology of proteins containing the beta-propeller domain could further elucidate the different pathways that the parasite could use to attain resistance.


Assuntos
Resistência a Medicamentos , Patrimônio Genético , Proteínas dos Microfilamentos/genética , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antimaláricos/farmacologia , Artemisininas/farmacologia , Repetição Kelch , Proteínas dos Microfilamentos/química , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/química
14.
BMC Res Notes ; 13(1): 497, 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33109270

RESUMO

OBJECTIVE: Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1-61 years old from South-west Nigeria. RESULTS: Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.


Assuntos
Combinação Arteméter e Lumefantrina/uso terapêutico , ATPases Transportadoras de Cálcio/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Adolescente , Adulto , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Resistência a Medicamentos/genética , Feminino , Expressão Gênica , Genótipo , Humanos , Lactente , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Nigéria/epidemiologia , Pandemias/prevenção & controle , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle
15.
PLoS One ; 15(10): e0228514, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33091033

RESUMO

Coral disease outbreaks are expected to increase in prevalence, frequency and severity due to climate change and other anthropogenic stressors. This is especially worrying for the Caribbean branching coral Acropora palmata which has already seen an 80% decrease in cover primarily due to disease. Despite the importance of this keystone species, there has yet to be a characterization of its transcriptomic response to disease exposure. In this study we provide the first transcriptomic analysis of 12 A. palmata genotypes and their symbiont Symbiodiniaceae exposed to disease in 2016 and 2017. Year was the primary driver of gene expression variance for A. palmata and the Symbiodiniaceae. We hypothesize that lower expression of ribosomal genes in the coral, and higher expression of transmembrane ion transport genes in the Symbiodiniaceae indicate that a compensation or dysbiosis may be occurring between host and symbiont. Disease response was the second driver of gene expression variance for A. palmata and included a core set of 422 genes that were significantly differentially expressed. Of these, 2 genes (a predicted cyclin-dependent kinase 11b and aspartate 1-decarboxylase) showed negative Log2 fold changes in corals showing transmission of disease, and positive Log2 fold changes in corals showing no transmission of disease, indicating that these may be important in disease resistance. Co-expression analysis identified two modules positively correlated to disease exposure, one enriched for lipid biosynthesis genes, and the other enriched in innate immune genes. The hub gene in the immune module was identified as D-amino acid oxidase, a gene implicated in phagocytosis and microbiome homeostasis. The role of D-amino acid oxidase in coral immunity has not been characterized but could be an important enzyme for responding to disease. Our results indicate that A. palmata mounts a core immune response to disease exposure despite differences in the disease type and virulence between 2016 and 2017. These identified genes may be important for future biomarker development in this Caribbean keystone species.


Assuntos
Alveolados/genética , Antozoários/parasitologia , Perfilação da Expressão Gênica/veterinária , Imunidade Inata , Animais , Antozoários/genética , Antozoários/imunologia , Mudança Climática , Regulação da Expressão Gênica , Genótipo , Proteínas de Protozoários/genética , Proteínas Ribossômicas/genética , Simbiose
16.
Nat Commun ; 11(1): 5342, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093501

RESUMO

Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF1. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , Microscopia Crioeletrônica , Lipídeos de Membrana/química , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/classificação , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Proteínas/química , Proteínas/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/ultraestrutura , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/ultraestrutura
17.
PLoS Pathog ; 16(8): e1008131, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866196

RESUMO

Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites.


Assuntos
Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Malária/epidemiologia , Merozoítos/enzimologia , Plasmodium berghei/enzimologia , Proteínas Quinases/biossíntese , Proteínas de Protozoários/biossíntese , Esporozoítos/enzimologia , Animais , Eritrócitos/enzimologia , Eritrócitos/parasitologia , Feminino , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/parasitologia , Malária/patologia , Camundongos
18.
PLoS Pathog ; 16(8): e1008772, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866214

RESUMO

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.


Assuntos
Antígenos de Protozoários , Babesia bovis , Conversão Gênica/imunologia , Genoma de Protozoário/imunologia , Proteínas de Protozoários , Rad51 Recombinase , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Babesia bovis/genética , Babesia bovis/imunologia , DNA de Protozoário/genética , DNA de Protozoário/imunologia , Haploidia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Rad51 Recombinase/genética , Rad51 Recombinase/imunologia
19.
PLoS Pathog ; 16(9): e1008799, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898164

RESUMO

Professional antigen-presenting cells (APCs), like macrophages (Mϕs) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoMϕs. Both MoDCs and MoMϕs readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoMϕs instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoMϕs show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFNγ) production by antigen specific CD8+ T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Macrófagos/imunologia , Malária/imunologia , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Pele/imunologia , Esporozoítos/imunologia , Animais , Células Cultivadas , Feminino , Humanos , Macrófagos/parasitologia , Malária/parasitologia , Camundongos , Pele/parasitologia
20.
Nucleic Acids Res ; 48(17): 9660-9680, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890403

RESUMO

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.


Assuntos
Variação Antigênica , DNA Polimerase Dirigida por DNA/metabolismo , Telômero/genética , Trypanosoma brucei brucei/genética , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/genética , Segregação de Cromossomos , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Regulação da Expressão Gênica , Genoma de Protozoário , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Telômero/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
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