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1.
PLoS Negl Trop Dis ; 13(12): e0007651, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31856154

RESUMO

Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo. The identification of parasite proteins synthesised during starvation is key to unravelling the underlying molecular mechanisms facilitating adaptation to these conditions. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesised Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified over 250 starvation-responsive NSPs in the parasite. Our results show a starvation-specific increased relative abundance of several translation regulating and stress-responsive proteins in the parasite. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 2.47e-35) and peptide biosynthetic process (enrichment P value 4.84e-35) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find widespread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.


Assuntos
Adaptação Fisiológica , Leishmania mexicana/química , Leishmania mexicana/fisiologia , Proteoma/análise , Proteômica/métodos , Proteínas de Protozoários/biossíntese , Inanição
2.
Malar J ; 18(1): 197, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196098

RESUMO

BACKGROUND: Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. METHODS: Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. RESULTS: Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. CONCLUSIONS: These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.


Assuntos
Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Adolescente , Adulto , África , Idoso , Idoso de 80 Anos ou mais , Alelos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Análise em Microsséries , Pessoa de Meia-Idade , Plasmodium vivax/imunologia , Proteínas de Protozoários/genética , Análise de Sequência de RNA , Adulto Jovem
3.
Parasitol Res ; 118(5): 1493-1500, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847614

RESUMO

Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.


Assuntos
Doença de Chagas/transmissão , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Animais , Linhagem Celular Tumoral , Doença de Chagas/parasitologia , Células HeLa , Humanos , México , Camundongos , Proteínas de Protozoários/genética , América do Sul , Trypanosoma cruzi/classificação , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
4.
Protein J ; 38(2): 167-180, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30905022

RESUMO

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Leucil Aminopeptidase/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/microbiologia , Concentração de Íons de Hidrogênio , Cinética , Leucina/análogos & derivados , Leucina/química , Leucil Aminopeptidase/antagonistas & inibidores , Leucil Aminopeptidase/química , Leucil Aminopeptidase/isolamento & purificação , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Temperatura
5.
PLoS One ; 14(2): e0212231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753239

RESUMO

Koumine is a component of the Chinese medicinal herb Gelsemium elegans and is toxic to vertebrates. We used the ciliate Tetrahymena thermophila as a model to evaluate the toxic effects of this indole alkaloid in eukaryotic microorganisms. Koumine inhibited T. thermophila growth and viability in a dose-dependent manner. Moreover, this drug produced oxidative stress in T. thermophila cells and expressions of antioxidant enzymes were significantly elevated at high koumine levels (p < 0.05). Koumine also caused significant levels of apoptosis (p < 0.05) and induced DNA damage in a dose-dependent manner. Mitophagic vacuoles were present in cells indicating induction of autophagy by this drug. Expression of ATG7, MTT2/4, CYP1 and HSP70 as well as the MAP kinase pathway gene MPK1 and MPK3 were significantly altered after exposed to koumine. This study represents a preliminary toxicological evaluation of koumine in the single celled eukaryote T. thermophila.


Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA , Alcaloides Indólicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Tetrahymena thermophila/metabolismo , Apoptose/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Estresse Oxidativo/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética
6.
Int J Parasitol ; 49(2): 127-137, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30367864

RESUMO

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.


Assuntos
Babesia bovis/fisiologia , Adesão Celular , Células Endoteliais/parasitologia , Expressão Gênica , Proteínas de Protozoários/biossíntese , Fatores de Virulência/biossíntese , Animais , Babesia bovis/genética , Bovinos , Células Cultivadas , Proteínas de Protozoários/genética , Fatores de Virulência/genética
7.
Sci Rep ; 8(1): 17566, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514885

RESUMO

Galactofuranosyltransferases are poorly described enzymes despite their crucial role in the virulence and the pathogenicity of numerous microorganisms. These enzymes are considered as potential targets for therapeutic action. In addition to the only well-characterised prokaryotic GlfT2 from Mycobacterium tuberculosis, four putative genes in Leishmania major were previously described as potential galactofuranosyltransferases. In this study, we have cloned, over-expressed, purified and fully determined the kinetic parameters of these four eukaryotic enzymes, thus demonstrating their unique potency in catalysing the transfer of the galactofuranosyl moiety into acceptors. Their individual promiscuity revealed to be different, as some of them could efficiently use NDP-pyranoses as donor substrates in addition to the natural UDP-galactofuranose. Such results pave the way for the development of chemoenzymatic synthesis of furanosyl-containing glycoconjugates as well as the design of improved drugs against leishmaniasis.


Assuntos
Galactose/análogos & derivados , Galactosiltransferases/biossíntese , Galactosiltransferases/química , Leishmania major/enzimologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Difosfato de Uridina/análogos & derivados , Biocatálise , Escherichia coli/genética , Galactose/metabolismo , Cinética , Especificidade por Substrato , Difosfato de Uridina/metabolismo
8.
Sci Rep ; 8(1): 17175, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30464192

RESUMO

Dinoflagellates are a diverse group of unicellular primary producers and grazers that exhibit some of the most remarkable features known among eukaryotes. These include gigabase-sized nuclear genomes, permanently condensed chromosomes and highly reduced organelle DNA. However, the genetic inventory that allows dinoflagellates to thrive in diverse ecological niches is poorly characterised. Here we systematically assess the functional capacity of 3,368,684 predicted proteins from 47 transcriptome datasets spanning eight dinoflagellate orders. We find that 1,232,023 proteins do not share significant sequence similarity to known sequences, i.e. are "dark". Of these, we consider 441,006 (13.1% of overall proteins) that are found in multiple taxa, or occur as alternative splice variants, to comprise the high-confidence dark proteins. Even with unknown function, 43.3% of these dark proteins can be annotated with conserved structural features using an exhaustive search against available data, validating their existence and importance. Furthermore, these dark proteins and their putative homologs are largely lineage-specific and recovered in multiple taxa. We also identified conserved functions in all dinoflagellates, and those specific to toxin-producing, symbiotic, and cold-adapted lineages. Our results demonstrate the remarkable divergence of gene functions in dinoflagellates, and provide a platform for investigations into the diversification of these ecologically important organisms.


Assuntos
Sequência Conservada , Dinoflagelados/genética , Genes de Protozoários , Genoma de Protozoário , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Conformação Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
9.
Protein Expr Purif ; 152: 122-130, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30059744

RESUMO

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30-75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg-1 plant material after purification based on an initial yield of ∼49 mg kg-1 biomass after blanching.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Antiprotozoários/química , Vacinas Antimaláricas/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Tabaco/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Cromatografia de Afinidade/métodos , Clonagem Molecular , Análise Fatorial , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Ligação Proteica , Desnaturação Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sefarose/análogos & derivados , Tabaco/química , Tabaco/metabolismo , Ultrafiltração/métodos
10.
Int J Biol Macromol ; 116: 1128-1145, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29782976

RESUMO

The pathogenicity of protozoan parasites is frequently attributed to their ability to circumvent the deleterious effects of ROS and Fe-S clusters are among their susceptible targets with paramount importance for parasite survival. The biogenesis of Fe-S clusters is orchestrated by ISC system; the sulfur donor IscS and scaffold protein IscU being its core components. However, among protozoan parasites including Leishmania, no information is available regarding biochemical aspect of IscU, its interaction partners and regulation. Here, we show that Leishmania donovani IscU homolog, LdIscU, readily assembles [2Fe-2S] clusters and, interestingly, follows Michaelis-Menten enzyme kinetics. It is localized in the mitochondria of the parasite and interacts with LdIscS to form a stable complex. Additionally, LdIscU and Fe-S proteins activity is significantly upregulated in resistant isolates and during stationary growth stage indicating an association between them. The differential expression of LdIscU modulated by Fe-S proteins demand suggests its potential role in parasite survival and drug resistance. Thus, our study provides novel insight into the Fe-S scaffold protein of a protozoan parasite.


Assuntos
Resistência a Medicamentos , Regulação da Expressão Gênica , Proteínas com Ferro-Enxofre/biossíntese , Leishmania donovani/metabolismo , Proteínas de Protozoários/biossíntese , Animais , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/genética , Leishmania donovani/química , Leishmania donovani/genética , Masculino , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Coelhos
11.
Parasitol Res ; 117(7): 2255-2263, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29779048

RESUMO

Interleukin 27 (IL-27) is a member of the IL-6/IL-12 family, and IL-27 receptor (IL-27R) consists of WSX-1 (the IL-27Rα subunit) and the signal-transducing subunit gp130. Human and mouse mast cells (MCs) express the IL-27R. To explore the expressions of IL-27/IL-27R subunits (WSX-1 and gp130) during acute ocular toxoplasmosis (OT), we established mouse model by intraocular injection of 500 Toxoplasma gondii RH strain tachyzoites. Histopathological changes were analyzed, MCs were counted by toluidine blue staining, and tryptase+/IL-27+ MCs were examined by immunofluorescence double-staining in the eyes and cervical lymph nodes (CLNs) of T. gondii-infected mice. The mRNA expressions of IL-27p28, WSX-1, gp130, and tachyzoite specific surface antigen 1 (SAG1) in the eyes and CLNs of T. gondii-infected mice, and the expressions of WSX-1 and gp130 in the murine mastocytoma cell line P815 infected with T. gondii tachyzoites in vitro were examined by using quantitative real-time reverse transcription-polymerase chain reaction. Our results showed that, after T. gondii infection, severe histopathological changes, increased numbers of total MCs and degranulated MCs, elevated expressions of IL-27p28, WSX-1, and gp130 were found in the eyes and CLNs, and significant correlations between the levels of IL-27 and SAG1 existed in the eyes and CLNs of T. gondii-infected mice. In addition, increased levels of WSX-1 and gp130 were examined in T. gondii-infected P815 cells. Our data suggested that IL-27/IL-27R expression induced by T. gondii infection may regulate MC-mediated immune response during acute OT in mouse model.


Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucinas/metabolismo , Mastócitos/metabolismo , Receptores de Citocinas/metabolismo , Toxoplasmose Ocular/patologia , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Receptor gp130 de Citocina/genética , Modelos Animais de Doenças , Feminino , Humanos , Interleucinas/genética , Mastócitos/imunologia , Mastocitoma/metabolismo , Camundongos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , RNA Mensageiro/biossíntese , Receptores de Citocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasmose Ocular/imunologia , Toxoplasmose Ocular/parasitologia
12.
Microb Cell Fact ; 17(1): 55, 2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29618355

RESUMO

BACKGROUND: The production of recombinant proteins with proper conformation, appropriate post-translational modifications in an easily scalable and cost-effective system is challenging. Lactococcus lactis has recently been identified as an efficient Gram positive cell factory for the production of recombinant protein. We and others have used this expression host for the production of selected malaria vaccine candidates. The safety of this production system has been confirmed in multiple clinical trials. Here we have explored L. lactis cell factories for the production of 31 representative Plasmodium falciparum antigens with varying sizes (ranging from 9 to 90 kDa) and varying degree of predicted structural complexities including eleven antigens with multiple predicted structural disulfide bonds, those which are considered difficult-to-produce proteins. RESULTS: Of the 31 recombinant constructs attempted in the L. lactis expression system, the initial expression efficiency was 55% with 17 out of 31 recombinant gene constructs producing high levels of secreted recombinant protein. The majority of the constructs which failed to produce a recombinant protein were found to consist of multiple intra-molecular disulfide-bonds. We found that these disulfide-rich constructs could be produced in high yields when genetically fused to an intrinsically disorder protein domain (GLURP-R0). By exploiting the distinct biophysical and structural properties of the intrinsically disordered protein region we developed a simple heat-based strategy for fast purification of the disulfide-rich protein domains in yields ranging from 1 to 40 mg/l. CONCLUSIONS: A novel procedure for the production and purification of disulfide-rich recombinant proteins in L. lactis is described.


Assuntos
Lactococcus lactis/metabolismo , Plasmodium falciparum/química , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes/biossíntese , Dissulfetos/química , Expressão Gênica , Plasmodium falciparum/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação
13.
J Cell Sci ; 131(9)2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29661850

RESUMO

The translationally controlled tumor protein (TCTP; also known as TPT1 in mammals) is highly conserved and ubiquitously expressed in eukaryotes. It is involved in growth and development, cell cycle progression, protection against cellular stresses and apoptosis, indicating the multifunctional role of the protein. Here, for the first time, we characterize the expression and function of TCTP in the human and animal pathogen, Trypanosoma brucei We identified two paralogs (TCTP1 and TCTP2) that are differentially expressed in the life cycle of the parasite. The genes have identical 5' untranslated regions (UTRs) and almost identical open-reading frames. The 3'UTRs differ substantially in sequence and length, and are sufficient for the exclusive expression of TCTP1 in procyclic- and TCTP2 in bloodstream-form parasites. Furthermore, we characterize which parts of the 3'UTR are needed for TCTP2 mRNA stability. RNAi experiments demonstrate that TCTP1 and TCTP2 expression is essential for normal cell growth in procyclic- and bloodstream-form parasites, respectively. Depletion of TCTP1 in the procyclic form cells leads to aberrant cell and mitochondrial organelle morphology, as well as enlarged, and a reduced number of, acidocalcisomes.


Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Regiões 3' não Traduzidas , Biomarcadores Tumorais/genética , Estágios do Ciclo de Vida/fisiologia , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
PLoS Negl Trop Dis ; 12(2): e0006220, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29432450

RESUMO

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase ß participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase ß To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase ß which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase ß were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase ß mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase ß in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.


Assuntos
DNA Polimerase beta/biossíntese , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia , Northern Blotting , Western Blotting , DNA Polimerase beta/metabolismo , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Peróxidos/toxicidade , Fosforilação , Proteoma/análise , Proteínas de Protozoários/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/efeitos dos fármacos
15.
Parasitol Res ; 117(4): 1095-1104, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29473141

RESUMO

Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans, has a complex life cycle that promotes survival in disparate environments. In each environment, the parasite must fine-tune its metabolic pathways to divide and multiply. In the absence of recognizable transcriptional gene regulation, it is apparent that protein levels are determined by post-transcriptional mechanisms. Post-transcriptional gene control is influenced by RNA-binding proteins that target mRNAs in the cell's cytoplasm. To initiate the study of post-transcriptional activities in T. cruzi, we studied this organism's ortholog of RBP42, a trypanosomal RNA-binding protein. RBP42 was originally detected in Trypanosoma brucei and was shown to target a subset of mRNAs that encode proteins governing central carbon metabolism. T. cruzi RBP42 structurally resembles T. brucei RBP42, sharing an NTF2 domain at its amino terminus and a single RNA-binding domain (specifically, the RNA recognition motif, or RRM), at its carboxy terminus. A phylogenetic analysis reveals that an NTF2 and a single RRM are distinguishing features of all RBP42 orthologs within the broad kinetoplastid grouping. T. cruzi RBP42 is expressed in all life cycle stages of the parasite as determined by immunoblot and immunofluorescence microscopy. In each case, the protein is localized to the cytoplasm, indicating a role for T. cruzi RBP42 in post-transcriptional activities in all stages of the parasite life cycle. We speculate that RBP42 influences the dynamic metabolic pathways responsible for parasite infection and transmission.


Assuntos
Proteínas de Protozoários/biossíntese , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/parasitologia , Citoplasma/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Estágios do Ciclo de Vida , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genética
16.
Dev Biol ; 435(1): 56-72, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29355521

RESUMO

GSK3 plays a central role in orchestrating key biological signaling pathways, including cell migration. Here, we identify GlkA as a GSK3 family kinase with functions that overlap with and are distinct from those of GskA. We show that GlkA, as previously shown for GskA, regulates the cell's cytoskeleton through MyoII assembly and control of Ras and Rap1 function, leading to aberrant cell migration. However, there are both qualitative and quantitative differences in the regulation of Ras and Rap1 and their downstream effectors, including PKB, PKBR1, and PI3K, with glkA- cells exhibiting a more severe chemotaxis phenotype than gskA- cells. Unexpectedly, the severe glkA- phenotypes, but not those of gskA-, are only exhibited when cells are grown attached to a substratum but not in suspension, suggesting that GlkA functions as a key kinase of cell attachment signaling. Using proteomic iTRAQ analysis we show that there are quantitative differences in the pattern of protein expression depending on the growth conditions in wild-type cells. We find that GlkA expression affects the cell's proteome during vegetative growth and development, with many of these changes depending on whether the cells are grown attached to a substratum or in suspension. These changes include key cytoskeletal and signaling proteins known to be essential for proper chemotaxis and signal relay during the aggregation stage of Dictyostelium development.


Assuntos
Quimiotaxia/fisiologia , Dictyostelium/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/biossíntese , Proteínas de Protozoários/biossíntese , Transdução de Sinais/fisiologia , Dictyostelium/genética , Quinase 3 da Glicogênio Sintase/genética , Proteínas de Protozoários/genética
17.
Biochim Biophys Acta Mol Cell Res ; 1865(11 Pt B): 1846-1856, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30992126

RESUMO

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca2+) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca2+ signaling is essential for the cellular and developmental changes that support T. gondii parasitism. The characterization of the molecular players and pathways directly activated by Ca2+ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca2+ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca2+ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Proteínas de Ligação ao Cálcio/metabolismo , Suscetibilidade a Doenças , Quinase 2 de Adesão Focal/metabolismo , Estágios do Ciclo de Vida , Proteínas de Protozoários/biossíntese
18.
PLoS One ; 13(12): e0206332, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30596656

RESUMO

The bloodstream form of the parasite Trypanosoma brucei obtains iron from its mammalian host by receptor-mediated endocytosis of host transferrin through its own unique transferrin receptor (TbTfR). Expression of TbTfR rapidly increases upon iron starvation by post-transcriptional regulation through a currently undefined mechanism that is distinct from the mammalian iron response system. We have created reporter cell lines by fusing the TbTfR 3'UTR or a control Aldolase 3'UTR to reporter genes encoding GFP or firefly Luciferase, and inserted the fusions into a bloodstream form cell line at a tagged ribosomal RNA locus. Fusion of the TbTfR 3'UTR is sufficient to significantly repress the expression of the reporter proteins under normal growth conditions. Under iron starvation conditions we observed upregulation of the mRNA and protein level of the TbTfR 3'UTR fusions only, with a magnitude and timing consistent with that reported for upregulation of the TbTfR. We conclude that the dynamic regulation of the T. brucei transferrin receptor in response to iron starvation is mediated via its 3'UTR, and that the effect is independent of genomic location.


Assuntos
Regiões 3' não Traduzidas , Regulação da Expressão Gênica , Ferro/deficiência , Proteínas de Protozoários/biossíntese , Receptores da Transferrina/biossíntese , Trypanosoma brucei brucei/metabolismo , Linhagem Celular , Humanos , Proteínas de Protozoários/genética , Receptores da Transferrina/genética , Trypanosoma brucei brucei/genética
19.
FEBS J ; 285(5): 848-870, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29281179

RESUMO

Transient regulation of Plasmodium numbers below the density that induces fever has been observed in chronic malaria infections in humans. This species transcending control cannot be explained by immunity alone. Using an in vitro system we have observed density dependent regulation of malaria population size as a mechanism to possibly explain these in vivo observations. Specifically, Plasmodium falciparum blood stages from a high but not low-density environment exhibited unique phenotypic changes during the late trophozoite (LT) and schizont stages of the intraerythrocytic cycle. These included in order of appearance: failure of schizonts to mature and merozoites to replicate, apoptotic-like morphological changes including shrinking, loss of mitochondrial membrane potential, and blebbing with eventual release of aberrant parasites from infected erythrocytes. This unique death phenotype was triggered in a stage-specific manner by sensing of a high-density culture environment. Conditions of glucose starvation, nutrient depletion, and high lactate could not induce the phenotype. A high-density culture environment induced rapid global changes in the parasite transcriptome including differential expression of genes involved in cell remodeling, clonal antigenic variation, metabolism, and cell death pathways including an apoptosis-associated metacaspase gene. This transcriptional profile was also characterized by concomitant expression of asexual and sexual stage-specific genes. The data show strong evidence to support our hypothesis that density sensing exists in P. falciparum. They indicate that an apoptotic-like mechanism may play a role in P. falciparum density regulation, which, as in yeast, has features quite distinguishable from mammalian apoptosis. DATABASE: Gene expression data are available in the GEO databases under the accession number GSE91188.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Malária Falciparum/parasitologia , Parasitemia/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Percepção de Quorum/genética , Transcrição Genética , Morte Celular , Meios de Cultura/farmacologia , Meios de Cultivo Condicionados/farmacologia , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Ontologia Genética , Humanos , Técnicas In Vitro , Malária Falciparum/sangue , Nutrientes/farmacologia , Fenótipo , Plasmodium falciparum/citologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética
20.
FASEB J ; 32(2): 906-919, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29046358

RESUMO

Translationally controlled tumor protein (TCTP) is a highly conserved, multifunctional protein that has been implicated in a range of cell physiologic processes, especially cell growth and development. A TCTP-like gene has been identified in the Toxoplasma genome [ Toxoplasma gondii TCTP ( TgTCTP)], although its function remains unknown. The sequence analysis of TgTCTP indicated that it is a highly conserved protein in eukaryotes. We found that the expression level of TgTCTP in the virulent RH strain was significantly higher than that in the avirulent PLK strain. Indirect immunofluorescence showed that TgTCTP was expressed in the parasite cytoplasm. The localization of TgTCTP was unchanged during the replication of the parasite. We expressed a functional recombinant TgTCTP (r TgTCTP) protein in Escherichia coli and found that the recombinant protein could form a multimer. We then evaluated the function of TgTCTP using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout (KO) system. Phenotypic analysis of the KO strain (Δ TgTCTP) revealed that TgTCTP is required for the robust growth of the parasites. TgTCTP deficiency also led to early egress of the parasites and subsequent impairment in their invasion and attachment abilities. We subsequently found that the multimer form of TgTCTP might not be necessary for the growth and replication of the parasite. Then the expression profiling of genes in the Δ TgTCTP and complement strains were analyzed. The results revealed that 988 genes were regulated in Δ TgTCTP compared with the complement strain. Overall, although not essential, TgTCTP is required for the fast growth of Tg and maintenance of its intracellular development.-Zheng, J., Chen, Y., Li, Z., Cao, S., Zhang, Z., Jia, H. Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.


Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Protozoários/biossíntese , Toxoplasma/crescimento & desenvolvimento , Biomarcadores Tumorais/genética , Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Protozoários/genética , Toxoplasma/genética
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