RESUMO
BACKGROUND: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. OBJECTIVES: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. METHODS: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. RESULTS: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. CONCLUSIONS: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmania tropica , Leishmaniose Visceral , Humanos , Feminino , Masculino , Cães , Animais , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/veterinária , Crithidia fasciculata , Irã (Geográfico)/epidemiologia , Doenças do Cão/epidemiologiaRESUMO
Kinetoplastid parasites are model eukaryotes with a complex cell cycle that is highly regulated both spatially and temporally. In addition, diseases caused by these parasites continue to have a significant impact on human and animal health worldwide. While there have been advancements in chemotherapy for these diseases, there is a continual need for an arsenal of compounds that have robust anti-parasite activity with minimal impact on the human host. While investigating a series of 2,3-diphenyl-2,3-dihydro-4H-1,3-thiaza-4-one heterocycles with potential activity against these parasites, we found a pyridothiazinone that inhibits growth of the monoxenous parasite Crithidia fasciculata and two life cycle stages of Trypanosoma brucei. This inhibition is more pronounced in T. brucei and is associated with an unusual pre-abscission cell cycle arrest. Exploring the mode of action for these and related compounds in kinetoplastids may provide tools with which to explore cell cycle regulation in these important organisms.
Assuntos
Parasitos , Trypanosoma brucei brucei , Animais , Compostos de Bifenilo , Crithidia fasciculata , Citocinese , HumanosRESUMO
Condensation and remodeling of nuclear genomes play an essential role in the regulation of gene expression and replication. Yet, our understanding of these processes and their regulatory role in other DNA-containing organelles, has been limited. This study focuses on the packaging of kinetoplast DNA (kDNA), the mitochondrial genome of kinetoplastids. Severe tropical diseases, affecting large human populations and livestock, are caused by pathogenic species of this group of protists. kDNA consists of several thousand DNA minicircles and several dozen DNA maxicircles that are linked topologically into a remarkable DNA network, which is condensed into a mitochondrial nucleoid. In vitro analyses implicated the replication protein UMSBP in the decondensation of kDNA, which enables the initiation of kDNA replication. Here, we monitored the condensation of kDNA, using fluorescence and atomic force microscopy. Analysis of condensation intermediates revealed that kDNA condensation proceeds via sequential hierarchical steps, where multiple interconnected local condensation foci are generated and further assemble into higher order condensation centers, leading to complete condensation of the network. This process is also affected by the maxicircles component of kDNA. The structure of condensing kDNA intermediates sheds light on the structural organization of the condensed kDNA network within the mitochondrial nucleoid.
Assuntos
Replicação do DNA/genética , DNA de Cinetoplasto/metabolismo , DNA Mitocondrial/genética , Núcleo Celular/metabolismo , Crithidia fasciculata/genética , DNA/metabolismo , DNA Circular/metabolismo , DNA de Cinetoplasto/genética , Proteínas de Ligação a DNA/genética , Genoma Mitocondrial/genética , Mitocôndrias/metabolismoRESUMO
Extracts of 35 samples of European propolis were tested against wild type and resistant strains of the protozoal pathogens Trypanosoma brucei, Trypanosoma congolense and Leishmania mexicana. The extracts were also tested against Crithidia fasciculata a close relative of Crithidia mellificae, a parasite of bees. Crithidia, Trypanosoma and Leishmania are all members of the order Kinetoplastida. High levels of activity were obtained for all the samples with the levels of activity varying across the sample set. The highest levels of activity were found against L. mexicana. The propolis samples were profiled by using liquid chromatography with high resolution mass spectrometry (LC-MS) and principal components analysis (PCA) of the data obtained indicated there was a wide variation in the composition of the propolis samples. Orthogonal partial least squares (OPLS) associated a butyrate ester of pinobanksin with high activity against T. brucei whereas in the case of T. congolense high activity was associated with methyl ethers of chrysin and pinobanksin. In the case of C. fasciculata highest activity was associated with methyl ethers of galangin and pinobanksin. OPLS modelling of the activities against L. mexicana using the mass spectrometry produced a less successful model suggesting a wider range of active components.
Assuntos
Antiprotozoários/farmacologia , Crithidia fasciculata/efeitos dos fármacos , Própole/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma congolense/efeitos dos fármacos , Animais , Antiprotozoários/uso terapêutico , Cromatografia Líquida , Infecções por Euglenozoa/tratamento farmacológico , Flavanonas/análise , Flavanonas/farmacologia , Flavonoides/análise , Flavonoides/farmacologia , Espectrometria de Massas , Própole/química , Própole/uso terapêuticoRESUMO
Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.
Assuntos
Aedes/parasitologia , Crithidia fasciculata/genética , Genes de Protozoários , Aedes/anatomia & histologia , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Crithidia fasciculata/crescimento & desenvolvimento , Crithidia fasciculata/fisiologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Masculino , RNA de Protozoário , Análise de Sequência de RNA , Transdução de SinaisRESUMO
Mitochondria are central organelles in cellular metabolism. Their structure is highly dynamic, allowing them to adapt to different energy requirements, to be partitioned during cell division, and to maintain functionality. Mitochondrial dynamics, including membrane fusion and fission reactions, are well studied in yeast and mammals but it is not known if these processes are conserved throughout eukaryotic evolution. Kinetoplastid parasites are some of the earliest-diverging eukaryotes to retain a mitochondrion. Each cell has only a single mitochondrial organelle, making them an interesting model for the role of dynamics in controlling mitochondrial architecture. We have investigated the mitochondrial division cycle in the kinetoplastid Crithidia fasciculata. The majority of mitochondrial biogenesis occurs during the G1 phase of the cell cycle, and the mitochondrion is divided symmetrically in a process coincident with cytokinesis. Live cell imaging revealed that the mitochondrion is highly dynamic, with frequent changes in the topology of the branched network. These remodeling reactions include tubule fission, fusion, and sliding, as well as new tubule formation. We hypothesize that the function of this dynamic remodeling is to homogenize mitochondrial contents and to facilitate rapid transport of mitochondria-encoded gene products from the area containing the mitochondrial nucleoid to other parts of the organelle.
Assuntos
Crithidia fasciculata/metabolismo , Fase G1/fisiologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Crithidia fasciculata/citologiaRESUMO
Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes. The screening assay had an average Z' factor of 0.7 and tolerated a maximum dimethyl sulfoxide concentration of up to 0.5%. We identified from multiple chemical boxes two compound series exhibiting nanomolar potency against C. fasciculata, one centred around a 5-nitrofuran-2-yl scaffold, a well-known moiety in several existing anti-infectives, and another involving a 2-(pyridin-2-yl) pyrimidin-4-amine scaffold which seems to have pan-kinetoplastid activity. This work facilitates the future use of C. fasciculata as a non-pathogenic and inexpensive biological resource to identify mode of action/protein target(s) of potentially pan-trypanocidal potent compounds. This knowledge will aid in the development of new treatments for African sleeping sickness, Chagas disease and leishmaniasis.
Assuntos
Antiprotozoários/farmacologia , Crithidia fasciculata/efeitos dos fármacos , Crithidia fasciculata/crescimento & desenvolvimento , Infecções por Euglenozoa/parasitologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Avaliação Pré-Clínica de Medicamentos , HumanosRESUMO
Kinetoplastid parasites are responsible for a range of diseases with significant global impact. Trypanosoma brucei and Trypanosoma cruzi cause human African trypanosomiasis and Chagas disease, respectively, while various Leishmania species are responsible for cutaneous, mucocutaneous and visceral leishmaniasis. Understanding the biology of these organisms is key for effective diagnosis, prophylaxis and treatment. The insect parasite Crithidia fasciculata offers a safe and low-cost alternative for studies of kinetoplastid biology. C. fasciculata does not infect humans, can be cultured to high yields in inexpensive serum-free medium in a standard laboratory, and has a completely sequenced publically available genome. Taking advantage of these features, however, requires the adaptation of existing methods of analysis to C. fasciculata. Tandem affinity purification is a widely used method that allows for the rapid purification of intact protein complexes under native conditions. Here we report the application of tandem affinity purification to C. fasciculata for the first time, demonstrating the effectiveness of the technique by purifying both the intact exosome and replication factor C complexes. Adding tandem affinity purification to the C. fasciculata toolbox significantly enhances the utility of this excellent model system.
Assuntos
Crithidia fasciculata/fisiologia , Infecções por Euglenozoa/parasitologia , Exossomos/metabolismo , Complexos Multiproteicos/isolamento & purificação , Proteína de Replicação C/isolamento & purificação , Proteína de Replicação C/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas Recombinantes de Fusão , Proteína de Replicação C/genética , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/metabolismo , Riboflavina/metabolismo , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Crithidia fasciculata/genética , Crithidia fasciculata/metabolismo , Humanos , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Estágios do Ciclo de Vida , Modelos Lineares , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Proteínas de Protozoários/genética , Ratos , Riboflavina/análogos & derivados , Trypanosoma cruzi/metabolismoRESUMO
Exoantigens (exo) from Leptomonas seymouri and Crithidia fasciculata were used in an enzyme linked immunosorbent assay (ELISA), showing 100% reactivity with sera from visceral leishmaniasis (VL) cases, and no reactivity with American tegumentary leishmaniasis (ATL) ones. Our results have indicated that these exoantigens can be applied in the discrimination of VL and ATL cases.
Assuntos
Antígenos de Protozoários/sangue , Crithidia fasciculata/imunologia , Leishmania donovani/imunologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Visceral/diagnóstico , Trypanosomatina/imunologia , Anticorpos Antiprotozoários/sangue , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologiaRESUMO
Exoantigens (exo) from Leptomonas seymouri and Crithidia fasciculata were used in an enzyme linked immunosorbent assay (ELISA), showing 100% reactivity with sera from visceral leishmaniasis (VL) cases, and no reactivity with American tegumentary leishmaniasis (ATL) ones. Our results have indicated that these exoantigens can be applied in the discrimination of VL and ATL cases
Assuntos
Humanos , Leishmaniose Cutânea/diagnóstico , Crithidia fasciculata , Trypanosomatina , Leishmaniose Visceral/diagnósticoRESUMO
We screened the genomes of a broad panel of kinetoplastid protists for genes encoding proteins associated with the RNA interference (RNAi) system using probes from the Argonaute (AGO1), Dicer1 (DCL1), and Dicer2 (DCL2) genes of Leishmania brasiliensis and Crithidia fasciculata. We identified homologs for all the three of these genes in the genomes of a subset of these organisms. However, several of these organisms lacked evidence for any of these genes, while others lacked only DCL2. The open reading frames encoding these putative proteins were structurally analyzed in silico. The alignments indicated that the genes are homologous with a high degree of confidence, and three-dimensional structural models strongly supported a functional relationship to previously characterized AGO1, DCL1, and DCL2 proteins. Phylogenetic analysis of these putative proteins showed that these genes, when present, evolved in parallel with other nuclear genes, arguing that the RNAi system genes share a common progenitor, likely across all Kinetoplastea. In addition, the genome segments bearing these genes are highly conserved and syntenic, even among those taxa in which they are absent. However, taxa in which these genes are apparently absent represent several widely divergent branches of kinetoplastids, arguing that these genes were independently lost at least six times in the evolutionary history of these organisms. The mechanisms responsible for the apparent coordinate loss of these RNAi system genes independently in several lineages of kinetoplastids, while being maintained in other related lineages, are currently unknown.
Assuntos
Crithidia fasciculata/genética , DNA de Cinetoplasto/genética , Leishmania braziliensis/genética , Trypanosomatina/genética , Sequência de Aminoácidos/genética , Proteínas Argonautas/genética , Evolução Biológica , DNA de Cinetoplasto/metabolismo , Eucariotos/genética , Evolução Molecular , Genoma/genética , Filogenia , Interferência de RNA/fisiologia , Ribonuclease III/genética , Alinhamento de Sequência/métodos , Sintenia/genéticaRESUMO
A glob is defined as a group of atoms in the crystal which can be chosen in various ways. Globs themselves can be used as scattering elements in the theory of structure determination, just as atoms are used at present. In this paper, amino-acid residues are chosen to form globs and empirical formulas for residue-based scattering factors have been developed.
Assuntos
Proteínas/química , Algoritmos , Aminoácidos/química , Proteínas de Bactérias/química , Crithidia fasciculata/química , Bases de Dados de Proteínas , Escherichia coli/química , Humanos , Análise dos Mínimos Quadrados , Conformação Proteica , Proteínas de Protozoários/química , Salmonella enterica/química , Thermus/química , Difração de Raios XRESUMO
Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 µg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 µg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.
Assuntos
Anti-Infecciosos/química , Antiprotozoários/química , Testes de Sensibilidade Microbiana , Própole/química , Animais , Anti-Infecciosos/farmacologia , Antiprotozoários/farmacologia , Produtos Biológicos/química , Cromatografia Líquida , Análise por Conglomerados , Crithidia fasciculata/efeitos dos fármacos , Feminino , Geografia , Humanos , Concentração Inibidora 50 , Análise dos Mínimos Quadrados , Leishmania donovani/efeitos dos fármacos , Líbia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium marinum/efeitos dos fármacos , Extratos Vegetais/química , Plasmodium falciparum/efeitos dos fármacos , Análise de Componente Principal , Própole/farmacologia , Software , Trypanosoma brucei brucei/efeitos dos fármacos , Células U937RESUMO
DNA topoisomerases are essential for DNA metabolism and while their role is well studied in prokaryotes and eukaryotes, it is less known for virally-encoded topoisomerases. African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA virus that infects Ornithodoros ticks and all members of the family Suidae, representing a global threat for pig husbandry with no effective vaccine nor treatment. It was recently demonstrated that ASFV codes for a type II topoisomerase, highlighting a possible target for control of the virus. In this work, the ASFV DNA topoisomerase II was expressed in Saccharomyces cerevisiae and found to efficiently decatenate kDNA and to processively relax supercoiled DNA. Optimal conditions for its activity were determined and its sensitivity to a panel of topoisomerase poisons and inhibitors was evaluated. Overall, our results provide new knowledge on viral topoisomerases and on ASFV, as well as a possible target for the control of this virus.
Assuntos
Vírus da Febre Suína Africana/enzimologia , DNA Topoisomerases Tipo II/genética , Inibidores da Topoisomerase II/farmacologia , Vírus da Febre Suína Africana/genética , Aminocumarinas/farmacologia , Amsacrina/farmacologia , Crithidia fasciculata/genética , Doxorrubicina/farmacologia , Saccharomyces cerevisiae/genéticaRESUMO
Kinetoplast DNA (kDNA), a unique mitochondrial structure common to trypanosomatid parasites, contains thousands of DNA minicircles that are densely packed and can be topologically linked into a chain mail-like network. Experimental data indicate that every minicircle in the network is, on average, singly linked to three other minicircles (i.e., has mean valence 3) before replication and to six minicircles in the late stages of replication. The biophysical factors that determine the topology of the network and its changes during the cell cycle remain unknown. Using a mathematical modeling approach, we previously showed that volume confinement alone can drive the formation of the network and that it induces a linear relationship between mean valence and minicircle density. Our modeling also predicted a minicircle valence two orders of magnitude greater than that observed in kDNA. To determine the factors that contribute to this discrepancy we systematically analyzed the relationship between the topological properties of the network (i.e., minicircle density and mean valence) and its biophysical properties such as DNA bending, electrostatic repulsion, and minicircle relative position and orientation. Significantly, our results showed that most of the discrepancy between the theoretical and experimental observations can be accounted for by the orientation of the minicircles with volume exclusion due to electrostatic interactions and DNA bending playing smaller roles. Our results are in agreement with the three dimensional kDNA organization model, initially proposed by Delain and Riou, in which minicircles are oriented almost perpendicular to the horizontal plane of the kDNA disk. We suggest that while minicircle confinement drives the formation of kDNA networks, it is minicircle orientation that regulates the topological complexity of the network.
Assuntos
Crithidia fasciculata/genética , DNA de Cinetoplasto/genética , DNA Mitocondrial/genética , Ciclo Celular/genética , Crithidia fasciculata/metabolismo , Replicação do DNA , DNA de Cinetoplasto/metabolismo , DNA Mitocondrial/metabolismoRESUMO
The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.
Assuntos
Cultura Axênica , Crithidia fasciculata/crescimento & desenvolvimento , Crithidia fasciculata/metabolismo , Leishmania/crescimento & desenvolvimento , Aglutinina de Amendoim/metabolismo , Proteômica , Aglutinação , Homeostase , Cinética , Metabolismo dos Lipídeos , Oxirredução , Via de Pentose Fosfato , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Especificidade da Espécie , Compostos de Sulfidrila/metabolismo , TranscriptomaRESUMO
Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.
Assuntos
Adesão Celular/efeitos dos fármacos , Crithidia fasciculata/efeitos dos fármacos , Crithidia fasciculata/crescimento & desenvolvimento , Glucose/metabolismo , Tunicamicina/farmacologia , Transporte Biológico/efeitos dos fármacos , Crithidia fasciculata/citologia , Crithidia fasciculata/metabolismo , Glicosilação/efeitos dos fármacosRESUMO
Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.
Assuntos
Adenosina/metabolismo , Crithidia fasciculata/metabolismo , Infecções por Euglenozoa/parasitologia , Proteínas de Transporte de Nucleosídeos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Clonagem Molecular , Crithidia fasciculata/química , Crithidia fasciculata/classificação , Crithidia fasciculata/genética , Humanos , Dados de Sequência Molecular , Proteínas de Transporte de Nucleosídeos/química , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Especificidade da EspécieRESUMO
BACKGROUND: Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe(3)O(4) MNPs in order to provoke cell death remotely using TVMFs. METHODS: Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO(4) in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry. RESULTS: MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. CONCLUSION: As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.