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1.
PLoS One ; 15(8): e0235401, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817665

RESUMO

BACKGROUND: Current malaria control and elimination strategies rely mainly on efficacious antimalarial drugs. However, drug resistance is a major threat facing malaria control programs. Determination of drug resistance molecular markers is useful in the monitoring and surveillance of malaria drug efficacy. This study aimed to determine the mutations and haplotypes frequencies of different genes linked with antimalarial drug resistance in certain areas in Sudan. METHODS: A total of 226 dried blood spots (DBS) of microscopically diagnosed P. falciparum isolates were collected from Khartoum and three other areas in Sudan during 2015-2017. Plasmodium falciparum confirmation and multiplicity of infection was assessed using the Sanger's 101 SNPs-barcode and speciation was confirmed using regions of the parasite mitochondria. Molecular genotyping of drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, exonuclease, Pfk13, parasite genetic background (PGB) (Pfarps10, ferredoxin, Pfcrt, Pfmdr2)) was also performed. All genotypes were generated by selective regions amplicon sequencing of the parasite genome using the Illumina MiSeq platform at the Wellcome Sanger Institute, UK then genotypes were translated into drug resistance haplotypes and species determination. FINDINGS: In total 225 samples were confirmed to be P. falciparum. A higher proportion of multiplicity of infection was observed in Gezira (P<0.001) based on the Sanger 101 SNPs -barcode. The overall frequency of mutant haplotype Pfcrt 72-76 CVIET was 71.8%. For Pfmdr1, N86Y was detected in 53.6%, Y184F was observed in 88.1% and D1246Y was detected in 1.5% of the samples. The most frequently observed haplotype was YFD 47.4%. For Pfdhfr (codons 51, 59,108,164), the ICNI haplotype was the most frequent (80.7%) while for Pfdhps (codons 436, 437, 540, 581, 613) the (SGEAA) was most frequent haplotype (41%). The Quadruple mutation (dhfr N51I, S108N + dhps A437G, K540E) was the highest frequent combined mutation (33.9%). In Pfkelch13 gene, 18 non-synonymous mutations were detected, 7 of them were detected in other African countries. The most frequent Pfk13 mutation was E433D detected in four samples. All of the Pfk13 mutant alleles have not been reported to belong to mutations associated with delayed parasite clearance in Southeast Asia. PGB mutations were detected only in Pfcrt N326S\I (46.3%) and Pfcrt I356T (8.2%). The exonuclease mutation was not detected. There was no significant variation in mutant haplotypes between study areas. CONCLUSIONS: There was high frequency of mutations in Pfcrt, Pfdhfr and Pfdhps in this study. These mutations are associated with chloroquine and sulfadoxine-pyrimethamine (SP) resistance. Many SNPs in Pfk13 not linked with delayed parasite clearance were observed. The exonuclease E415G mutation which is linked with piperaquine resistance was not reported.


Assuntos
Resistência a Medicamentos/genética , Malária/parasitologia , Mutação , Plasmodium falciparum/genética , Adolescente , Antimaláricos/farmacologia , Criança , Cloroquina/farmacologia , Feminino , Humanos , Malária/epidemiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirimetamina/farmacologia , Sudão , Sulfadoxina/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Adulto Jovem
2.
PLoS Biol ; 18(8): e3000774, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745097

RESUMO

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.


Assuntos
Dictyostelium/genética , MAP Quinase Quinase Quinase 3/genética , Proteínas de Protozoários/genética , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Animais , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular Tumoral , Quimiotaxia/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Edição de Genes/métodos , Regulação da Expressão Gênica , MAP Quinase Quinase Quinase 3/metabolismo , Melanócitos/metabolismo , Melanócitos/ultraestrutura , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Células NIH 3T3 , Fenótipo , Fosforilação , Ploidias , Proteínas de Protozoários/metabolismo , Pseudópodes/genética , Pseudópodes/ultraestrutura , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
3.
PLoS Biol ; 18(8): e3000756, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745139

RESUMO

Recognition of self and nonself is important for outcrossing organisms, and different mating types establish the barrier against self-mating. In the unicellular ciliate T. thermophila, mating type determination requires complex DNA rearrangements at a single mat locus during conjugation to produce a type-specific gene pair (MTA and MTB) for 1 of 7 possible mating types. Surprisingly, we found that decreased expression of the DNA breakage-repair protein Ku80 at late stages of conjugation generated persistent selfing phenotype in the progeny. DNA analysis revealed multiple mating-type gene pairs as well as a variety of mis-paired, unusually arranged mating-type genes in these selfers that resemble some proposed rearrangement intermediates. They are found also in normal cells during conjugation and are lost after 10 fissions but are retained in Ku mutants. Silencing of TKU80 or TKU70-2 immediately after conjugation also generated selfing phenotype, revealing a hidden DNA rearrangement process beyond conjugation. Mating reactions between the mutant and normal cells suggest a 2-component system for self-nonself-recognition through MTA and MTB genes.


Assuntos
DNA de Protozoário/genética , Rearranjo Gênico , Autoantígeno Ku/genética , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética , Conjugação Genética , Cruzamentos Genéticos , DNA de Protozoário/metabolismo , Expressão Gênica , Inativação Gênica , Autoantígeno Ku/metabolismo , Fenótipo , Proteínas de Protozoários/metabolismo , Reprodução , Tetrahymena thermophila/metabolismo
4.
PLoS Pathog ; 16(8): e1008810, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817704

RESUMO

Sterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm‾). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm‾ mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm‾ mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm‾. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.


Assuntos
Leishmania major/enzimologia , Leishmania major/fisiologia , Leishmaniose Cutânea/patologia , Mitocôndrias/fisiologia , Proteínas de Protozoários/metabolismo , Esterol 14-Desmetilase/metabolismo , Esteróis/metabolismo , Humanos , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Potencial da Membrana Mitocondrial , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio/metabolismo , Esterol 14-Desmetilase/genética
5.
Nat Commun ; 11(1): 3825, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732874

RESUMO

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.


Assuntos
Lipídeos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/parasitologia
6.
Nat Commun ; 11(1): 3922, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764664

RESUMO

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico Ativo , Resistência a Medicamentos/genética , Feminino , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Oócitos/metabolismo , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/genética , Xenopus laevis
7.
PLoS Pathog ; 16(8): e1008327, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853276

RESUMO

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Inflamassomos/imunologia , Interferon gama/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Linfócitos T CD8-Positivos/parasitologia , Feminino , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Protozoários/genética , Toxoplasmose Animal/parasitologia , Vacúolos/imunologia , Vacúolos/metabolismo , Vacúolos/parasitologia , Virulência/imunologia
8.
PLoS Negl Trop Dis ; 14(8): e0008509, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804927

RESUMO

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.


Assuntos
Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Leishmania/genética , Consumo de Oxigênio , Proteômica , Proteínas de Protozoários/genética , Especificidade da Espécie
9.
Nucleic Acids Res ; 48(14): 7899-7913, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609816

RESUMO

In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only are previously established modifications observable by mass spectrometry in strains with the most conserved genes of each step deleted, but also additional modifications are detected, indicating a certain plasticity of the pathway in the amoeba. Unlike described for yeast, D. discoideum allows for an unconditional deletion of the single tQCUG gene, as long as the Elongator-dependent modification pathway is intact. In gene deletion strains of the modification pathway, protein amounts are significantly reduced as shown by flow cytometry and Western blotting, using strains expressing different glutamine leader constructs fused to GFP. Most dramatic are these effects, when the tQCUG gene is deleted, or Elp3, the catalytic component of the Elongator complex is missing. In addition, Elp3 is the most strongly conserved protein of the modification pathway, as our phylogenetic analysis reveals. The implications of this observation are discussed with respect to the evolutionary age of the components acting in the Elongator-dependent modification pathway.


Assuntos
Dictyostelium/genética , RNA de Transferência/metabolismo , Anticódon/química , Anticódon/metabolismo , Códon , Dictyostelium/metabolismo , Deleção de Genes , Glutamina , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Mutação , Nucleosídeos/química , Filogenia , Biossíntese de Proteínas , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Uridina/metabolismo
10.
PLoS Pathog ; 16(7): e1008650, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628723

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.


Assuntos
Proteínas de Protozoários/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/metabolismo , Fatores de Virulência/metabolismo , Animais , Camundongos , Fosfolipases/genética , Fosfolipases/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/enzimologia , Virulência
11.
PLoS Negl Trop Dis ; 14(7): e0008439, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628683

RESUMO

Leishmaniasis constitutes the 9th largest disease burden among all infectious diseases. Control of this disease is based on a short list of chemotherapeutic agents headed by pentavalent antimonials, followed by miltefosine and amphotericin B; drugs that are far from ideal due to host toxicity, elevated cost, limited access, and high rates of drug resistance. Knowing that the composition of extracellular vesicles (EVs) can vary according to the state of their parental cell, we hypothesized that EVs released by drug-resistant Leishmania infantum parasites could contain unique and differently enriched proteins depending on the drug-resistance mechanisms involved in the survival of their parental cell line. To assess this possibility, we studied EV production, size, morphology, and protein content of three well-characterized drug-resistant L. infantum cell lines and a wild-type strain. Our results are the first to demonstrate that drug-resistance mechanisms can induce changes in the morphology, size, and distribution of L. infantum EVs. In addition, we identified L. infantum's core EV proteome. This proteome is highly conserved among strains, with the exception of a handful of proteins that are enriched differently depending on the drug responsible for induction of antimicrobial resistance. Furthermore, we obtained the first snapshot of proteins enriched in EVs released by antimony-, miltefosine- and amphotericin-resistant parasites. These include several virulence factors, transcription factors, as well as proteins encoded by drug-resistance genes. This detailed study of L. infantum EVs sheds new light on the potential roles of EVs in Leishmania biology, particularly with respect to the parasite's survival in stressful conditions. This work outlines a crucial first step towards the discovery of EV-based profiles capable of predicting response to antileishmanial agents.


Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/metabolismo , Biologia Computacional , Vesículas Extracelulares , Regulação da Expressão Gênica/fisiologia , Proteoma , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
12.
Nat Commun ; 11(1): 3532, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669539

RESUMO

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/enzimologia , Proteína Fosfatase 1/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Proliferação de Células , GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Fosfatidilcolinas/química , Domínios Proteicos , Proteoma , Ubiquitina-Proteína Ligases/metabolismo
13.
Parasitol Res ; 119(8): 2667-2678, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32627078

RESUMO

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).


Assuntos
Eimeria/citologia , Eimeria/ultraestrutura , Animais , Eimeria/crescimento & desenvolvimento , Glicoproteínas/química , Glicoproteínas/metabolismo , Estágios do Ciclo de Vida , Microscopia Eletrônica , Microscopia de Fluorescência , Oocistos/citologia , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Oocistos/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/química
14.
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
15.
Nat Commun ; 11(1): 2763, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488076

RESUMO

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.


Assuntos
Ritmo Circadiano/fisiologia , Eritropoese/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Malária/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Animais , Proteínas de Caenorhabditis elegans , Modelos Animais de Doenças , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/genética , Humanos , Malária/parasitologia , Camundongos , Camundongos Knockout , Plasmodium chabaudi/genética , Plasmodium chabaudi/crescimento & desenvolvimento , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Receptores Acoplados a Proteínas-G/genética , Roedores , Transcriptoma
16.
PLoS One ; 15(6): e0234918, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579605

RESUMO

ZapE/Afg1 is a component of the inner cell membrane of some eubacteria and the inner mitochondrial membrane of eukaryotes. This protein is involved in FtsZ-dependent division of eubacteria. In the yeast and human mitochondrion, ZapE/Afg1 likely interacts with Oxa1 and facilitates the degradation of mitochondrion-encoded subunits of respiratory complexes. Furthermore, the depletion of ZapE increases resistance to apoptosis, decreases oxidative stress tolerance, and impacts mitochondrial protein homeostasis. It remains unclear whether ZapE is a multifunctional protein, or whether some of the described effects are just secondary phenotypes. Here, we have analyzed the functions of ZapE in Trypanosoma brucei, a parasitic protist, and an important model organism. Using a newly developed proximity-dependent biotinylation approach (BioID2), we have identified the inner mitochondrial membrane insertase Oxa1 among three putative interacting partners of ZapE, which is present in two paralogs. RNAi-mediated depletion of both ZapE paralogs likely affected the function of respiratory complexes I and IV. Consistently, we show that the distribution of mitochondrial ZapE is restricted only to organisms with Oxa1, respiratory complexes, and a mitochondrial genome. We propose that the evolutionarily conserved interaction of ZapE with Oxa1, which is required for proper insertion of many inner mitochondrial membrane proteins, is behind the multifaceted phenotype caused by the ablation of ZapE.


Assuntos
Deleção de Genes , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Biotinilação , Regulação para Baixo , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eucariotos/genética , Genoma Mitocondrial , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fenótipo , Filogenia , Ligação Proteica
17.
Nat Commun ; 11(1): 3228, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591529

RESUMO

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.


Assuntos
5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Biocatálise , Plasmodium falciparum/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Mutantes/química , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Especificidade por Substrato
18.
PLoS Pathog ; 16(6): e1008455, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32544189

RESUMO

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.


Assuntos
Ciclo Celular , Enzimas Desubiquitinantes/metabolismo , Leishmania mexicana/enzimologia , Proteínas de Protozoários/metabolismo , Ubiquitinação , Animais , Enzimas Desubiquitinantes/genética , Feminino , Deleção de Genes , Leishmania mexicana/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética
19.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
20.
PLoS Pathog ; 16(6): e1008640, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569299

RESUMO

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.


Assuntos
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Ubiquitina/genética
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