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1.
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
2.
Molecules ; 26(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672916

RESUMO

The in vitro activity of L. donovani (promastigotes, axenic amastigotes and intracellular amastigotes in THP1 cells) and T. brucei, from the fractions obtained from the hydroalcoholic extract of the aerial part of Hypericum afrum and the isolated compounds, has been evaluated. The chloroform, ethyl acetate and n-butanol extracts showed significant antitrypanosomal activity towards T. brucei, with IC50 values of 12.35, 13.53 and 12.93 µg/mL and with IC90 values of 14.94, 19.31 and 18.67 µg/mL, respectively. The phytochemical investigation of the fractions led to the isolation and identification of quercetin (1), myricitrin (2), biapigenin (3), myricetin (4), hyperoside (5), myricetin-3-O-ß-d-galactopyranoside (6) and myricetin-3'-O-ß-d-glucopyranoside (7). Myricetin-3'-O-ß-d-glucopyranoside (7) has been isolated for the first time from this genus. The chemical structures were elucidated by using comprehensive one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopic data, as well as high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). These compounds have also been evaluated for their antiprotozoal activity. Quercetin (1) and myricetin (4) showed noteworthy activity against T. brucei, with IC50 and IC90 values of 7.52 and 5.71 µM, and 9.76 and 7.97 µM, respectively. The T. brucei hexokinase (TbHK1) enzyme was further explored as a potential target of quercetin and myricetin, using molecular modeling studies. This proposed mechanism assists in the exploration of new candidates for novel antitrypanosomal drugs.


Assuntos
Antiprotozoários/farmacologia , Flavonoides/farmacologia , Hypericum/química , Modelos Moleculares , Compostos Fitoquímicos/farmacologia , Quercetina/farmacologia , Trypanosoma/efeitos dos fármacos , Sequência de Aminoácidos , Antiprotozoários/química , Sítios de Ligação , Morte Celular/efeitos dos fármacos , Sequência Conservada , Flavonoides/química , Flavonoides/isolamento & purificação , Ligantes , Simulação de Dinâmica Molecular , Compostos Fitoquímicos/química , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Quercetina/química , Quercetina/isolamento & purificação , Água/química
3.
Nat Commun ; 12(1): 1750, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741942

RESUMO

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterize B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230 kDa gamete surface protein Pfs230, a key protein in sexual stage development of P. falciparum parasites. Among nine Pfs230 human monoclonal antibodies (mAbs) that we generated, one potently blocks transmission to mosquitoes in a complement-dependent manner and reacts to the gamete surface; the other eight show only low or no blocking activity. The structure of the transmission-blocking mAb in complex with vaccine antigen reveals a large discontinuous conformational epitope, specific to domain 1 of Pfs230 and comprising six structural elements in the protein. The epitope is conserved, suggesting the transmission-blocking mAb is broadly functional. This study provides a rational basis to improve malaria vaccines and develop therapeutic antibodies for malaria elimination.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antiprotozoários/farmacologia , Epitopos/imunologia , Células Germinativas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Adulto , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Sítios de Ligação , Células Cultivadas , Epitopos/química , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interações Hospedeiro-Parasita/imunologia , Humanos , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Mosquitos Vetores/parasitologia , Plasmodium falciparum/imunologia , Plasmodium falciparum/fisiologia , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia
4.
Nat Commun ; 12(1): 1538, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750786

RESUMO

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.


Assuntos
Anticorpos Bloqueadores , Anticorpos Monoclonais/imunologia , Proteínas de Membrana/metabolismo , Plasmodium vivax/metabolismo , Proteínas de Protozoários/metabolismo , Reticulócitos/metabolismo , Anticorpos Antiprotozoários/imunologia , Antígenos CD , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Camboja , Cristalografia por Raios X , Humanos , Estudos Longitudinais , Malária Vivax/imunologia , Malária Vivax/parasitologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Plasmodium vivax/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Receptores da Transferrina
5.
Science ; 371(6532): 910-916, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33632841

RESUMO

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliate Tetrahymena to identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo-electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.


Assuntos
Dineínas do Axonema/metabolismo , Cílios/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/fisiologia , Dineínas do Axonema/química , Dineínas do Axonema/genética , Microscopia Crioeletrônica , Citoplasma/metabolismo , Técnicas de Silenciamento de Genes , Processamento de Imagem Assistida por Computador , Microtúbulos/fisiologia , Modelos Moleculares , Movimento , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética
6.
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
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.
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
10.
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
11.
Parasitol Res ; 119(9): 3013-3022, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32740752

RESUMO

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.


Assuntos
Proteínas de Artrópodes/metabolismo , Babesia microti/enzimologia , Cistatinas/metabolismo , Cisteína Proteases/metabolismo , Proteínas de Protozoários/metabolismo , Carrapatos/metabolismo , Carrapatos/parasitologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Babesia bovis/química , Babesia bovis/enzimologia , Babesia bovis/genética , Babesia microti/química , Babesia microti/genética , Babesiose/parasitologia , Cistatinas/genética , Cisteína Proteases/química , Cisteína Proteases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Carrapatos/genética
12.
Parasitol Res ; 119(9): 2991-3003, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32748038

RESUMO

Visceral leishmaniasis (VL, also known as kala-azar) is a vector borne disease caused by obligate intracellular protozoan parasite Leishmania donovani. To overcome the limitations of currently available drugs for VL, molecular target-based study is a promising tool to develop new drugs to treat this neglected tropical disease. One such target we recently identified from L. donovani (Ld) genome (WGS, clinical Indian isolate; BHU 1220, AVPQ01000001) is a small GTP-binding protein, Rab6 protein. We now report a specific inhibitor of the GTPase activity of Rab6 protein of L. donovani (LdRab6) without restricting host enzyme activity. First, to understand the nature of LdRab6 protein, we generated recombinant LdRab6 mutant proteins (rLdRab6) by systematically introducing deletion (two cysteine residues at C-terminal) and mutations [single amino acid substitutions in the conserved region of GTP (Q84L)/GDP(T38N) coding sequence]. The GTPase activity of rLdRab6:GTP and rLdRab6:GDP locked mutant proteins showed ~ 8-fold and ~ 1.5-fold decreases in enzyme activity, respectively, compared to the wild type enzyme activity. The mutant protein rLdRab6:ΔC inhibited the GTPase activity. Sequence alignment analysis of Rab6 protein of L. donovani with Homo sapiens showed identical amino acids in the G conserved region (GTP/GDP-binding sites) but it differed in the C-terminal region. We then evaluated the inhibitory activity of trans-dibenzalacetone (DBA, a synthetic analog of curcumin with strong antileishmanial activity reported earlier by us) in the GTPase activity of LdRab6 protein. Comparative molecular docking analysis of DBA and specific inhibitors of Rab proteins (Lovastatin, BFA, Zoledronate, and NE10790) indicated that DBA had optimum binding affinity with LdRab6 protein. This was further confirmed by the GTPase activity of DBA-treated LdRab6 which showed a basal GTP level significantly lower than that of the wild-type rLdRab6. The results confirm that DBA inhibits the GTPase activity of LdRab6 protein from L. donovani (LdRab6), a potential target for its antileishmanial effect.


Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Pentanonas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Curcumina/farmacologia , Humanos , Leishmania donovani/química , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leishmaniose Visceral/tratamento farmacológico , Simulação de Acoplamento Molecular , Pentanonas/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
13.
Exp Parasitol ; 218: 107967, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32858044

RESUMO

Reported incidence rates of cryptosporidiosis in Ireland are consistently among the highest in Europe. Despite the national prevalence of this enteric parasite and the compulsory nature of incidence surveillance and reporting, in-depth analyses seeking to genotype clinical isolates of Cryptosporidium on an intra-species level are rarely undertaken in Ireland. This molecular epidemiology study of 163 clinical Cryptosporidium isolates was conducted in Southern Ireland, from 2015 to 2018, in order to ascertain population subtype heterogeneity. Analysis was conducted via real-time PCR amplification and gp60 gene sequencing, which successfully determined the subtype designation of 149 of the 163 (91.4%) tested isolates. Overall, 12 C. parvum and five C. hominis subtypes were identified, with the incidence of the regionally predominant C. parvum species found to primarily occur during springtime months, while C. hominis incidence was largely confined to late summer and autumnal months. Additionally, one C. parvum and four C. hominis subtypes were newly reported by this study, having not been previously identified in clinical or livestock infection in Ireland. Overall, these data give insight into the diversification of the Cryptosporidium population and emergent subtypes, while also allowing comparisons to be made with clinical epidemiological profiles reported previously in Ireland and elsewhere.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/classificação , Zoonoses/parasitologia , Animais , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Gastroenterite/parasitologia , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Incidência , Irlanda/epidemiologia , Estudos Longitudinais , Prevalência , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Estações do Ano , Alinhamento de Sequência
14.
PLoS Negl Trop Dis ; 14(7): e0008488, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716931

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. CONCLUSION: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.


Assuntos
Doenças do Cão/diagnóstico , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Doenças do Cão/sangue , Cães , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Transcriptoma
15.
Proc Natl Acad Sci U S A ; 117(29): 16790-16798, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631977

RESUMO

Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.


Assuntos
Aptâmeros de Nucleotídeos/química , L-Lactato Desidrogenase/química , Malária/diagnóstico , Proteínas de Protozoários/química , Biomarcadores/sangue , Biomarcadores/química , Humanos , Ligação de Hidrogênio , L-Lactato Desidrogenase/sangue , Malária/sangue , Técnicas de Diagnóstico Molecular/métodos , Simulação de Dinâmica Molecular , Plasmodium vivax/enzimologia , Ligação Proteica
16.
PLoS Negl Trop Dis ; 14(7): e0008202, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645098

RESUMO

Plasmodium vivax is the most widespread and difficult to treat cause of human malaria. The development of vaccines against the blood stages of P. vivax remains a key objective for the control and elimination of vivax malaria. Erythrocyte binding-like (EBL) protein family members such as Duffy binding protein (PvDBP) are of critical importance to erythrocyte invasion and have been the major target for vivax malaria vaccine development. In this study, we focus on another member of EBL protein family, P. vivax erythrocyte binding protein (PvEBP). PvEBP was first identified in Cambodian (C127) field isolates and has subsequently been showed its preferences for binding reticulocytes which is directly inhibited by antibodies. We analysed PvEBP sequence from 316 vivax clinical isolates from eight countries including China (n = 4), Ethiopia (n = 24), Malaysia (n = 53), Myanmar (n = 10), Papua New Guinea (n = 16), Republic of Korea (n = 10), Thailand (n = 174), and Vietnam (n = 25). PvEBP gene exhibited four different phenotypic clusters based on the insertion/deletion (indels) variation. PvEBP-RII (179-479 aa.) showed highest polymorphism similar to other EBL family proteins in various Plasmodium species. Whereas even though PvEBP-RIII-V (480-690 aa.) was the most conserved domain, that showed strong neutral selection pressure for gene purifying with significant population expansion. Antigenicity of both of PvEBP-RII (16.1%) and PvEBP-RIII-V (21.5%) domains were comparatively lower than other P. vivax antigen which expected antigens associated with merozoite invasion. Total IgG recognition level of PvEBP-RII was stronger than PvEBP-RIII-V domain, whereas total IgG inducing level was stronger in PvEBP-RIII-V domain. These results suggest that PvEBP-RII is mainly recognized by natural IgG for innate protection, whereas PvEBP-RIII-V stimulates IgG production activity by B-cell for acquired immunity. Overall, the low antigenicity of both regions in patients with vivax malaria likely reflects genetic polymorphism for strong positive selection in PvEBP-RII and purifying selection in PvEBP-RIII-V domain. These observations pose challenging questions to the selection of EBP and point out the importance of immune pressure and polymorphism required for inclusion of PvEBP as a vaccine candidate.


Assuntos
Variação Genética , Malária Vivax/imunologia , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Anticorpos Antiprotozoários/imunologia , Ásia , Humanos , Imunidade Humoral , Malária Vivax/parasitologia , Plasmodium vivax/química , Plasmodium vivax/imunologia , Polimorfismo Genético , Proteínas de Protozoários/química , Seleção Genética , Alinhamento de Sequência
17.
Nucleic Acids Res ; 48(15): 8645-8662, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32614436

RESUMO

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.


Assuntos
Polinucleotídeo Adenililtransferase/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Poliadenilação/genética , Proteínas de Protozoários/química , Edição de RNA/genética , Precursores de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Protozoário/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
18.
Proc Natl Acad Sci U S A ; 117(28): 16546-16556, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601225

RESUMO

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.


Assuntos
Heme/metabolismo , Lipocalinas/metabolismo , Malária/parasitologia , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Hemeproteínas/genética , Hemeproteínas/metabolismo , Humanos , Lipocalinas/química , Lipocalinas/genética , Malária/metabolismo , Camundongos , Plasmodium berghei/química , Plasmodium berghei/genética , Plasmodium falciparum/química , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
19.
Exp Parasitol ; 216: 107941, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622940

RESUMO

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Medula Óssea/parasitologia , Biologia Computacional , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Imunoglobulina G/sangue , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas de Protozoários/química , Sensibilidade e Especificidade , Alinhamento de Sequência , Testes Sorológicos , Baço/parasitologia , Adulto Jovem
20.
Arch Biochem Biophys ; 690: 108468, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679196

RESUMO

Hsp90 is a ubiquitous, homodimer and modular molecular chaperone. Each Hsp90 protomer has three different domains, named the N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD). The Hsp90 molecular cycle involves ATP binding and hydrolysis, which drive conformational changes. Hsp90 is critical for the viability of eukaryotic organisms, including the protozoan that causes the severe form of malaria, Plasmodium falciparum, the growth and differentiation of which are compromised when Hsp90 is inhibited. Here, we characterize the structure of a recombinant P. falciparum Hsp90 (PfHsp90) protein, as well as its MD (PfHsp90MD) and NTD plus MD (PfHsp90NMD) constructs. All the proteins were obtained with high purity and in the folded state. PfHsp90 and PfHsp90NMD interacted with adenosine nucleotides via the NTD, and Mg2+ was critical for strong binding. PfHsp90 behaved mostly as elongated and flexible dimers in solution, which dissociate with a sub-micromolar dissociation constant. The PfHsp90MD and PfHsp90NMD constructs behaved as globular and elongated monomers, respectively, confirming the importance of the CTD for dimerization. Small angle X-ray scattering data were obtained for all the constructs, and ab initio models were constructed, revealing PfHsp90 in an open conformation and as a greatly elongated and flexible protein.


Assuntos
Proteínas de Choque Térmico HSP90/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Adenosina/química , Trifosfato de Adenosina/química , Sítios de Ligação , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Hidrólise , Magnésio/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica
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