RESUMO
Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.
Assuntos
Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Regulação da Expressão Gênica , RNA de Protozoário/biossíntese , Animais , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Entamebíase/genética , Entamebíase/terapia , Humanos , Camundongos , RNA de Protozoário/genética , RatosRESUMO
Introducción. Los mecanismos de resistencia al antimonio pentavalente conocidos hasta el momento, se han descrito ampliamente en cepas del subgénero Leishmania, pero poco se sabe sobre las proteínas involucradas en los mecanismos de resistencia presentes en cepas del subgénero Viannia, como Leishmania panamensis. Objetivo. Identificar proteínas diferencialmente expresadas entre las cepas de L. panamensis (UA140), sensible y resistente al antimonio pentavalente, y analizar el posible papel de estas proteínas en mecanismos de resistencia. Materiales y métodos. Las proteínas de las cepas, sensible y resistente al antimonio pentavalente, se compararon usando electroforesis bidimensional. Las proteínas con aumento de la expresión fueron aisladas e identificadas por espectrometría de masas mediante MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). La expresión del ARNm de cinco de estas proteínas se cuantificó mediante PCR en tiempo real. Resultados. Los geles bidimensionales de las cepas sensible y resistente detectaron 532±39 y 541±43 manchas proteicas. Se encontraron 10 manchas con aumento de la expresión en la cepa resistente, identificadas como proteínas de choque térmico (Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica), isomerasa de disulfuro, proteasa de cisteína, enolasa, factor de elongación 5-α, la subunidad 5-α del proteasoma y dos proteínas hipotéticas nombradas como Sp(2) y Sp(25). Conclusión. Este es el primer estudio llevado a cabo con una cepa resistente al antimonio pentavalente en L. panamensis, en el cual se han identificado proteínas que están relacionadas con el mecanismo de resistencia del parásito frente al medicamento, abriendo el camino para futuros estudios de estas proteínas como blancos terapéuticos.
Introduction. The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. Objective. Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. Materials and methods. The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. Results. On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). Conclusion. This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.
Assuntos
Antiprotozoários/farmacologia , Técnicas In Vitro , Leishmania guyanensis/metabolismo , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Proteínas de Protozoários/biossíntese , Resistência a Medicamentos , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Leishmania guyanensis/efeitos dos fármacos , Leishmania guyanensis/genética , Proteômica , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Técnica de SubtraçãoRESUMO
INTRODUCTION: The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. OBJECTIVE: Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. MATERIALS AND METHODS: The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. RESULTS: On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). CONCLUSION: This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.
Assuntos
Antiprotozoários/farmacologia , Leishmania guyanensis/metabolismo , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Proteínas de Protozoários/biossíntese , Resistência a Medicamentos , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Técnicas In Vitro , Leishmania guyanensis/efeitos dos fármacos , Leishmania guyanensis/genética , Antimoniato de Meglumina , Proteômica , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Técnica de SubtraçãoRESUMO
Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.
Assuntos
Deleção de Genes , Perfilação da Expressão Gênica , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Proteínas de Transporte de Monossacarídeos/deficiência , Proteoma/análise , Proteínas de Protozoários/análise , Eletroforese em Gel Bidimensional , Immunoblotting , Espectrometria de Massas , Análise em Microsséries , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients. METHODS: The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts. RESULTS: 7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates. CONCLUSION: Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis.
Assuntos
Leishmania/isolamento & purificação , Leishmania/fisiologia , Leishmaniose Cutânea/parasitologia , Viabilidade Microbiana , Reação em Cadeia da Polimerase/métodos , Sobrevivência Celular , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Monócitos/parasitologia , Tonsila Palatina/parasitologia , Parasitologia/métodos , RNA de Protozoário/biossíntese , RNA de Protozoário/genética , RNA Citoplasmático Pequeno/biossíntese , RNA Citoplasmático Pequeno/genética , Partícula de Reconhecimento de Sinal/biossíntese , Partícula de Reconhecimento de Sinal/genética , Pele/parasitologiaRESUMO
Differential gene expression in three pairs of Trypanosoma cruzi populations or clones susceptible or resistant to benznidazole (BZ) was investigated by differential display (DD) and representation of differential expression (RDE). GenBank searches of 14 genes selected by DD showed that four sequences corresponded to different hypothetical proteins and the others were very similar to T. cruzi genes encoding mucin (TcMUC), dihydrolipoamide dehydrogenase (TcLipDH), the hexose transporter (TcHT), or a ribosomal protein. Sequence analysis was performed on 34 clones obtained by RDE; approximately half of these clones encoded 14 different hypothetical proteins and the other half encoded proteins involved with stress response, antioxidant defence, metabolism, transporter proteins, surface proteins, ribosomal proteins and others. The mRNA levels of eight T. cruzi genes obtained by RDE and DD were analysed by northern blotting to confirm the differential expression of these sequences. For six of the eight genes, TcLipDH, TcHT, TcFeSOD-A (iron superoxide dismutase-A), TcHSP70, TcHSP100 (heat shock protein) and Tc52 (thiol-transferase), mRNA levels in the drug-resistant T. cruzi population were at least twice those in the susceptible population. Further analysis of TcHSP70 showed that although the levels of TcHSP70 mRNA were four-fold higher in T. cruzi BZ-resistant population, no corresponding increase was observed in the levels of TcHSP70 protein expression. The results suggest that TcHSP70 is not directly associated with the T. cruzi drug resistance phenotype.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Perfilação da Expressão Gênica , Nitroimidazóis/farmacologia , Trypanosoma cruzi/genética , Animais , Northern Blotting , DNA de Protozoário/química , DNA de Protozoário/genética , Genes de Protozoários , Dados de Sequência Molecular , Proteínas de Protozoários/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA de Protozoário/biossíntese , RNA de Protozoário/genética , Análise de Sequência de DNA , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The metabolism of pentose phosphates was studied in Leishmania mexicana promastigotes. Each of the enzymes of the classical pentose phosphate pathway (PPP) has been identified and specific activities measured. Functioning of the PPP was demonstrated in non-growing cells by measuring the evolution of 14CO2 from [1-14C]D-glucose and [6-14C]D-glucose under normal conditions and also under selective stimulation of the PPP by exposure to methylene blue. The proportion of glucose which passes through the PPP increases in the latter condition, thus suggesting a protective role against oxidant stress. The incorporation into nucleic acids of ribose 5-phosphate provided via either glucose or free ribose was also determined. Results indicate that the PPP enables glucose to serve as a source of ribose 5-phosphate in nucleotide biosynthesis. Moreover, free ribose is incorporated efficiently, implying the presence of a ribose uptake system and also of ribokinase. Ribose was shown to be accumulated by a carrier mediated process in L. mexicana promastigotes and ribokinase activity was also measured in these cells.
Assuntos
Leishmania mexicana/metabolismo , Via de Pentose Fosfato , Animais , Transporte Biológico , Dióxido de Carbono/metabolismo , Radioisótopos de Carbono , Proteínas de Transporte/metabolismo , DNA de Protozoário/biossíntese , Glucose/metabolismo , Hidrólise , Leishmania mexicana/enzimologia , Azul de Metileno/metabolismo , Azul de Metileno/farmacologia , Ácidos Nucleicos/biossíntese , Nucleosídeos/metabolismo , Estresse Oxidativo/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA de Protozoário/biossíntese , Ribose/metabolismo , Ribosemonofosfatos/metabolismoRESUMO
The effects of juvenile hormone-III (JH-III) and the juvenile hormone analogues (JHA) methoprene and fenoxycarb on the growth and macromolecular biosynthesis in Trypanosoma cruzi were studied in vitro. It was observed that JH-III and JHA blocked growth and 3H-thymidine incorporation without killing the cells within certain concentrations (< or = 1 x 10(-4) M), but they caused cellular death at concentrations over 1 x 10(-3) M. The inhibitory effect on growth was partially reversible. On the other hand, the inhibitory action of JH-III, methoprene and fenoxycarb was an unspecific effect according to the results obtained with Leishmania mexicana mexicana (promastigotes) and human peripheral blood lymphocytes. The JHA have a good possibility of being used in the control of trypanosomiasis.
Assuntos
Carbamatos/farmacologia , Hormônios Juvenis/farmacologia , Leishmania mexicana/efeitos dos fármacos , Metoprene/farmacologia , Fenilcarbamatos , Trypanosoma cruzi/efeitos dos fármacos , Animais , DNA de Protozoário/biossíntese , DNA de Protozoário/efeitos dos fármacos , Humanos , Inseticidas/farmacologia , Leishmania mexicana/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/parasitologia , Biossíntese de Proteínas , Precursores de Proteínas/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Proteínas/efeitos dos fármacos , Precursores de RNA/efeitos dos fármacos , Precursores de RNA/metabolismo , RNA de Protozoário/biossíntese , RNA de Protozoário/efeitos dos fármacos , Timidina/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Mucins are heavily O-glycosylated Thr/Ser/Pro-rich molecules. Given their relevant functions, mucins and their genes have been mainly studied in higher eukaryotes. In the protozoan parasite Trypanosoma cruzi, mucin-like glycoproteins were shown to play an important role in the interaction with the surface of the mammalian cell during the invasion process. We show now that this parasite has a family of putative mucin genes, whose organization resembles the one present in mammalian cells. Different parasite isolates have different sets of genes, as defined by their central domain. Central domains, rich in codons for Thr and/or Ser and Pro residues, are made up of either a variable number of repeat units in tandem or non-repetitive sequences. Conversely, 5'- and 3'-ends from different genes in different isolates have similar sequences, suggesting their common origin. Comparison of deduced amino acid sequences revealed that all members of the family have the same putative signal peptide on the N terminus and a putative sequence for glycophosphatidylinositol anchoring on the C terminus. The deduced molecular mass of the core proteins is small (from 17 to 21 kDa), in agreement with the 1-kilobase size of the mRNA detected. Putative mucin genes in T. cruzi are located on large chromosomal bands of about 1.6-2.2 megabase pairs.
Assuntos
Genes de Protozoários , Mamíferos/genética , Mucinas/genética , Família Multigênica , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Chlorocebus aethiops , Clonagem Molecular , Códon , Glicosilfosfatidilinositóis/biossíntese , Dados de Sequência Molecular , Mucinas/biossíntese , Mucinas/química , Oligodesoxirribonucleotídeos , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/química , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , RNA de Protozoário/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Serina , Especificidade da Espécie , Treonina , Células VeroRESUMO
Sinefungin and its cyclic analog were evaluated in vitro for activity against the multiplication of Trypanosoma cruzi. When either drug was tested for eight days on twelve T. cruzi epimastigote isolates, an 800-fold difference in drug sensitivity was found. Both drugs were trypanostatics at a concentration range from 0.1 micrograms/ml to 300 micrograms/ml. The 50% effective concentration (EC50) of sinefungin and its cyclic analog at which the growth of a given isolate was inhibited was 0.38 micrograms/ml for sinefungin and 0.31 micrograms/ml for the cyclic analog against the Ma, Marin, OPS-86, Y, and Ya isolates, and > 300 micrograms/ml for sinefungin and 217 micrograms/ml for the cyclic analog against the A-35, Bertoldo, DS, EP, ES, OPS-58, and FL isolates. Incubation of drug-sensitive isolates for more than 10 days in glucose-saline (GS) medium, but not in minimal essential medium, in the presence of a 30-fold EC50 concentration of the drug induced an increase in the drug-resistant population, which maintained this phenotype for several passages in drug-free culture medium. Growth curves were analyzed as a function of parasite inoculum; it was observed that with sinefungin-sensitive T. cruzi epimastigote isolates grown in GS medium in the presence of 10 micrograms/ml of the drug, the inhibitory effects of the drug were dependent on the initial inoculum: 1 x 10(3)-1 x 10(4) parasites/ml were strongly inhibited even after 16 days. Significant impairment of thymidine incorporation into the DNA of parasites by both drugs was observed only in drug-sensitive epimastigote isolates.(ABSTRACT TRUNCATED AT 250 WORDS)