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1.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206906

RESUMO

Leishmania survival inside macrophages depends on factors that lead to the immune response evasion during the infection. In this context, the metabolic scenario of the host cell-parasite relationship can be crucial to understanding how this parasite can survive inside host cells due to the host's metabolic pathways reprogramming. In this work, we aimed to analyze metabolic networks of bone marrow-derived macrophages from C57BL/6 mice infected with Leishmania amazonensis wild type (La-WT) or arginase knocked out (La-arg-), using the untargeted Capillary Electrophoresis-Mass Spectrometry (CE-MS) approach to assess metabolomic profile. Macrophages showed specific changes in metabolite abundance upon Leishmania infection, as well as in the absence of parasite-arginase. The absence of L. amazonensis-arginase promoted the regulation of both host and parasite urea cycle, glycine and serine metabolism, ammonia recycling, metabolism of arginine, proline, aspartate, glutamate, spermidine, spermine, methylhistidine, and glutathione metabolism. The increased L-arginine, L-citrulline, L-glutamine, oxidized glutathione, S-adenosylmethionine, N-acetylspermidine, trypanothione disulfide, and trypanothione levels were observed in La-WT-infected C57BL/6-macrophage compared to uninfected. The absence of parasite arginase increased L-arginine, argininic acid, and citrulline levels and reduced ornithine, putrescine, S-adenosylmethionine, glutamic acid, proline, N-glutamyl-alanine, glutamyl-arginine, trypanothione disulfide, and trypanothione when compared to La-WT infected macrophage. Moreover, the absence of parasite arginase leads to an increase in NO production levels and a higher infectivity rate at 4 h of infection. The data presented here show a host-dependent regulation of metabolomic profiles of C57BL/6 macrophages compared to the previously observed BALB/c macrophages infected with L. amazonensis, an important fact due to the dual and contrasting macrophage phenotypes of those mice. In addition, the Leishmania-arginase showed interference with the urea cycle, glycine, and glutathione metabolism during host-pathogen interactions.


Assuntos
Aminoácidos/metabolismo , Interações Hospedeiro-Parasita , Leishmaniose/metabolismo , Macrófagos/metabolismo , Metaboloma , Poliaminas/metabolismo , Animais , Arginase/metabolismo , Células Cultivadas , Leishmania/enzimologia , Leishmania/patogenicidade , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas de Protozoários/metabolismo
2.
Front Cell Infect Microbiol ; 11: 704662, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34268141

RESUMO

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.


Assuntos
Aquaporinas , Esporozoítos , Animais , Hepatócitos/metabolismo , Humanos , Plasmodium falciparum , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Tetraspanina 28/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204357

RESUMO

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.


Assuntos
Alveolados/fisiologia , Apicomplexa/metabolismo , Diatomáceas/metabolismo , Heme/metabolismo , Redes e Vias Metabólicas , Sequência de Aminoácidos , Transporte Biológico , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
4.
Nat Commun ; 12(1): 4226, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244481

RESUMO

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.


Assuntos
Antígenos de Protozoários/ultraestrutura , Malária Falciparum/imunologia , Proteínas de Membrana/ultraestrutura , Plasmodium falciparum/imunologia , Proteínas de Protozoários/ultraestrutura , Receptores Imunológicos/ultraestrutura , Anticorpos Antiprotozoários/genética , Anticorpos Antiprotozoários/metabolismo , Variação Antigênica/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Cristalografia por Raios X , Humanos , Malária Falciparum/parasitologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Mutação , Plasmodium falciparum/metabolismo , Domínios Proteicos/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo
5.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202520

RESUMO

Trypanosoma brucei (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against T. brucei.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/classificação , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/metabolismo , Citosol/metabolismo , Imunofluorescência , Proteínas de Choque Térmico HSP70/química , Azul de Metileno/química , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Proteínas de Protozoários/química , Quercetina/química , Coloração e Rotulagem , Relação Estrutura-Atividade
6.
Nat Commun ; 12(1): 3788, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145271

RESUMO

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.


Assuntos
Membrana Celular/parasitologia , Proteínas de Protozoários/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/patologia , Transporte Biológico/fisiologia , Células Cultivadas , Interações Hospedeiro-Parasita , Humanos , Via Secretória/fisiologia , Fatores de Virulência
7.
Nat Commun ; 12(1): 3820, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155201

RESUMO

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.


Assuntos
Estágios do Ciclo de Vida , Mitocôndrias/metabolismo , Plasmodium falciparum/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Evolução Molecular , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Fosforilação Oxidativa , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestrutura , Especificidade da Espécie
8.
Nat Commun ; 12(1): 3620, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131147

RESUMO

Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp. induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles. To understand this process and address the debate regarding how the parasitophorous vacuole membrane (PVM) is formed, we developed an approach using lattice light-sheet microscopy, which enables the parasite interaction with the host cell membrane to be tracked and characterized during invasion. Our results show that the PVM is predominantly formed from the erythrocyte membrane, which undergoes biophysical changes as it is remodeled across all stages of invasion, from pre-invasion through to PVM sealing. This approach enables a functional interrogation of parasite-derived lipids and proteins in PVM biogenesis and echinocytosis during Plasmodium falciparum invasion and promises to yield mechanistic insights regarding how this is more generally orchestrated by other intracellular pathogens.


Assuntos
Membrana Eritrocítica/parasitologia , Eritrócitos/parasitologia , Tomografia Computadorizada Quadridimensional/métodos , Interações Hospedeiro-Parasita/fisiologia , Malária/parasitologia , Vacúolos/metabolismo , Animais , Membrana Eritrocítica/metabolismo , Humanos , Merozoítos , Parasitos , Plasmodium/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
9.
Nucleic Acids Res ; 49(11): 6196-6212, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086947

RESUMO

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.


Assuntos
Chaperonas de Histonas/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Evolução Biológica , Sequência Conservada , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Expressão Gênica , Células HEK293 , Chaperonas de Histonas/química , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidade , Oncogenes , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/crescimento & desenvolvimento
10.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071922

RESUMO

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.


Assuntos
Entamoeba histolytica/fisiologia , Entamebíase/metabolismo , Entamebíase/parasitologia , Interações Hospedeiro-Parasita , Fagocitose , Proteínas de Protozoários/metabolismo , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismo , Sítios de Ligação , Citofagocitose , Entamoeba histolytica/classificação , Entamebíase/imunologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Genoma de Protozoário , Humanos , Modelos Moleculares , Fagossomos , Filogenia , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sumoilação
11.
FASEB J ; 35(7): e21685, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34085343

RESUMO

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca2+ and K+ homeostasis in mammalian cells. Here, we demonstrate that the Letm1 orthologue of Trypanosoma cruzi, the etiologic agent of Chagas disease, is important for mitochondrial Ca2+ uptake and release. The results show that both mitochondrial Ca2+ influx and efflux are reduced in TcLetm1 knockdown (TcLetm1-KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential. Remarkably, TcLetm1 knockdown or overexpression increases or does not affect mitochondrial Ca2+ levels in epimastigotes, respectively. TcLetm1-KD epimastigotes have reduced growth, and both overexpression and knockdown of TcLetm1 cause a defect in metacyclogenesis. TcLetm1-KD also affected mitochondrial bioenergetics. Invasion of host cells by TcLetm1-KD trypomastigotes and their intracellular replication is greatly impaired. Taken together, our findings indicate that TcLetm1 is important for Ca2+ homeostasis and cell viability in T cruzi.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Doença de Chagas/parasitologia , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Transporte Biológico , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Chlorocebus aethiops , Metabolismo Energético , Potencial da Membrana Mitocondrial , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Trypanosoma cruzi/metabolismo , Células Vero
12.
Molecules ; 26(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34066964

RESUMO

The serine protease, DegP exhibits proteolytic and chaperone activities, essential for cellular protein quality control and normal cell development in eukaryotes. The P. falciparum DegP is essential for the parasite survival and required to combat the oscillating thermal stress conditions during the infection, protein quality checks and protein homeostasis in the extra-cytoplasmic compartments, thereby establishing it as a potential target for drug development against malaria. Previous studies have shown that diisopropyl fluorophosphate (DFP) and the peptide SPMFKGV inhibit E. coli DegP protease activity. To identify novel potential inhibitors specific to PfDegP allosteric and the catalytic binding sites, we performed a high throughput in silico screening using Malaria Box, Pathogen Box, Maybridge library, ChEMBL library and the library of FDA approved compounds. The screening helped identify five best binders that showed high affinity to PfDegP allosteric (T0873, T2823, T2801, RJC02337, CD00811) and the catalytic binding site (T0078L, T1524, T2328, BTB11534 and 552691). Further, molecular dynamics simulation analysis revealed RJC02337, BTB11534 as the best hits forming a stable complex. WaterMap and electrostatic complementarity were used to evaluate the novel bio-isosteric chemotypes of RJC02337, that led to the identification of 231 chemotypes that exhibited better binding affinity. Further analysis of the top 5 chemotypes, based on better binding affinity, revealed that the addition of electron donors like nitrogen and sulphur to the side chains of butanoate group are more favoured than the backbone of butanoate group. In a nutshell, the present study helps identify novel, potent and Plasmodium specific inhibitors, using high throughput in silico screening and bio-isosteric replacement, which may be experimentally validated.


Assuntos
Antimaláricos/farmacologia , Simulação por Computador , Desenho de Fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Antimaláricos/química , Sítios de Ligação , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Evolução Molecular , Simulação de Acoplamento Molecular , Peptídeos/química , Peptídeos/farmacologia , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Eletricidade Estática , Termodinâmica , Água/química
13.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068436

RESUMO

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote's EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.


Assuntos
Doença de Chagas/metabolismo , Vesículas Extracelulares/metabolismo , Estágios do Ciclo de Vida , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/parasitologia , Chlorocebus aethiops , Vesículas Extracelulares/parasitologia , Interações Hospedeiro-Parasita , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteoma/análise , Células Vero
14.
Nat Commun ; 12(1): 2947, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011950

RESUMO

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.


Assuntos
Evolução Molecular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Modelos Moleculares , Naegleria/classificação , Naegleria/genética , Naegleria/metabolismo , Peroxissomos/metabolismo , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/classificação
15.
Front Cell Infect Microbiol ; 11: 656620, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937099

RESUMO

Erythrocyte recognition and invasion is critical for the intra-erythrocytic development of Plasmodium spp. parasites. The multistep invasion process involves specific interactions between parasite ligands and erythrocyte receptors. Erythrocyte-binding-like (EBL) proteins, type I integral transmembrane proteins released from the merozoite micronemes, are known to play an important role in the initiation and formation of tight junctions between the apical end of the merozoite and the erythrocyte surface. In Plasmodium yoelii EBL (PyEBL), a single amino acid substitution in the putative Duffy binding domain dramatically changes parasite growth rate and virulence. This suggests that PyEBL is important for modulating the virulence of P. yoelii parasites. Based on these observations, we sought to elucidate the receptor of PyEBL that mediates its role as an invasion ligand. Using the eukaryotic wheat germ cell-free system, we systematically developed and screened a library of mouse erythrocyte proteins against native PyEBL using AlphaScreen technology. We report that PyEBL specifically interacts with basigin, an erythrocyte surface protein. We further confirmed that the N-terminal cysteine-rich Duffy binding-like region (EBL region 2), is responsible for the interaction, and that the binding is not affected by the C351Y mutation, which was previously shown to modulate virulence of P. yoelii. The identification of basigin as the putative PyEBL receptor offers new insights into the role of this molecule and provides an important base for in-depth studies towards developing novel interventions against malaria.


Assuntos
Plasmodium yoelii , Animais , Antígenos de Protozoários/metabolismo , Basigina , Eritrócitos , Proteínas de Membrana , Camundongos , Plasmodium falciparum , Plasmodium yoelii/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo
16.
Gene ; 792: 145723, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34019936

RESUMO

Avian malaria is a common and widespread disease of birds caused by a diverse group of pathogens of the genera Plasmodium. We investigated the transcriptomal profiles of one of the most common species, Plasmodium relictum, lineage SGS1, at multiple timepoints during the blood stages of the infection under experimental settings. The parasite showed well separated overall transcriptome profiles between day 8 and 20 after the infection, shown by well separated PCA profiles. Moreover, gene expression becomes more heterogenous within the experimental group late in the infection, either due to adaptations to individual differences between the experimental hosts, or due to desynchronisation of the life-cycle of the parasite. Overall, this study shows how the avian malaria system can be used to study gene expression of the avian Plasmodium parasite under controlled experimental settings, thus allowing for future comparative analysis of gene responses of parasite with different life-history traits and host effects.


Assuntos
Estágios do Ciclo de Vida/genética , Malária Aviária/parasitologia , Plasmodium/genética , Proteínas de Protozoários/genética , Transcriptoma , Animais , Aves/parasitologia , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Ontologia Genética , Anotação de Sequência Molecular , Filogenia , Plasmodium/classificação , Plasmodium/crescimento & desenvolvimento , Plasmodium/metabolismo , Análise de Componente Principal , Proteínas de Protozoários/classificação , Proteínas de Protozoários/metabolismo
17.
FASEB J ; 35(6): e21641, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34041791

RESUMO

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket ("big eye" phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.


Assuntos
Endocitose , Lisossomos/metabolismo , Organelas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/fisiologia , Tripanossomíase/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Transporte Proteico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Interferência de RNA , Tripanossomíase/parasitologia , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/genética
18.
Nat Commun ; 12(1): 2813, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001876

RESUMO

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.


Assuntos
Membrana Celular/metabolismo , Lipidômica/métodos , Fosfatidato Fosfatase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Silenciamento de Genes , Homeostase/genética , Interações Hospedeiro-Parasita , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fosfatidato Fosfatase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/ultraestrutura
19.
PLoS Negl Trop Dis ; 15(4): e0009284, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909626

RESUMO

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.


Assuntos
Membrana Basal/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Meios de Cultura , Estágios do Ciclo de Vida , Proteínas de Protozoários/metabolismo , Percepção de Quorum , Trypanosoma brucei brucei/citologia
20.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925823

RESUMO

The development of novel anti-infectives against Kinetoplastids pathogens targeting proteins is a big problem occasioned by the antigenic variation in these parasites. This is also a global concern due to the zoonosis of these parasites, as they infect both humans and animals. Therefore, we need not only to create novel antibiotics, but also to speed up the development pipeline for these antibiotics. This may be achieved by using novel drug targets for Kinetoplastids drug discovery. In this study, we focused our attention on motifs of rRNA molecules that have been created using homology modeling. The RNA is the most ambiguous biopolymer in the kinetoplatid, which carries many different functions. For instance, tRNAs, rRNAs, and mRNAs are essential for gene expression both in the pro-and eukaryotes. However, all these types of RNAs have sequences with unique 3D structures that are specific for kinetoplastids only and can be used to shut down essential biochemical processes in kinetoplastids only. All these features make RNA very potent targets for antibacterial drug development. Here, we combine in silico methods combined with both computational biology and structure prediction tools to address our hypothesis. In this study, we outline a systematic approach for identifying kinetoplastid rRNA-ligand interactions and, more specifically, techniques that can be used to identify small molecules that target particular RNA. The high-resolution optimized model structures of these kineoplastids were generated using RNA 123, where all the stereochemical conflicts were solved and energies minimized to attain the best biological qualities. The high-resolution optimized model's structures of these kinetoplastids were generated using RNA 123 where all the stereochemical conflicts were solved and energies minimized to attain the best biological qualities. These models were further analyzed to give their docking assessment reliability. Docking strategies, virtual screening, and fishing approaches successfully recognized novel and myriad macromolecular targets for the myxobacterial natural products with high binding affinities to exploit the unmet therapeutic needs. We demonstrate a sensible exploitation of virtual screening strategies to 18S rRNA using natural products interfaced with classical maximization of their efficacy in phamacognosy strategies that are well established. Integration of these virtual screening strategies in natural products chemistry and biochemistry research will spur the development of potential interventions to these tropical neglected diseases.


Assuntos
Produtos Biológicos/química , Kinetoplastida/genética , RNA Ribossômico 18S/química , Animais , Doença de Chagas/tratamento farmacológico , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Genes de Protozoários , Humanos , Kinetoplastida/metabolismo , Kinetoplastida/patogenicidade , Leishmaniose/tratamento farmacológico , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mapeamento de Interação de Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/metabolismo , Tripanossomíase/tratamento farmacológico
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