RESUMO
In this work, we characterized the gene encoding iron superoxide dismutase-A (FeSOD-A) in wild-type (WTS) and antimony-resistant (SbR) L. (Viannia) braziliensis and L. (Leishmania) infantum lines, which were selected in vitro. FeSOD-A transcript and protein expression were similar in all tested lines; however, specific enzyme activity analysis revealed higher superoxide dismutase activity in SbIII-resistant LbSbR and LiSbR lines than in the corresponding WTS lines. These parasites were also more tolerant to oxidative stress generated by the herbicide paraquat. Functional analysis showed that in comparison to non-transfected lines, wild-type LbWTS and LiWTS clones overexpressing the FeSOD-A enzyme are 1.6- and 1.7-fold more resistant to SbIII, respectively. Our results suggest that FeSOD-A is involved in the antimony resistance phenotype in L. (V.) braziliensis and L. (L.) infantum.
Assuntos
Antimônio/farmacologia , Resistência a Medicamentos , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Superóxido Dismutase/classificação , Superóxido Dismutase/metabolismo , Antiprotozoários/farmacologia , Regulação Enzimológica da Expressão Gênica , Herbicidas/farmacologia , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Paraquat/farmacologia , Superóxido Dismutase/genéticaRESUMO
The antioxidant system in plants is a very important defensive mechanism to overcome stress conditions. We examined the expression profile of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) using a bioinformatics approach. We explored secondary structure prediction and made detailed studies of signature pattern of antioxidant proteins in four plant species (Triticum aestivum, Arabidopsis thaliana, Oryza sativa, and Brassica juncea). Fingerprinting analysis was done with ScanProsite, which includes a large collection of biologically meaningful signatures. Multiple sequence alignment of antioxidant proteins of the different plant species revealed a conserved secondary structure region, indicating homology at the sequence and structural levels. The secondary structure prediction showed that these proteins have maximum tendency for α helical structure. The sequence level similarities were also analyzed with a phylogenetic tree using neighbor-joining method. In the antioxidant enzymes SOD, CAT and APX, three major families of signature were predominant and common; these were PKC_PHOSPHO_SITE, CK2_PHOSPHO_SITE and N-myristoylation site, which are functionally related to various plant signaling pathways. This study provides new strategies for screening of biomodulators involved in plant stress metabolism that will be useful for designing degenerate primers or probes specific for antioxidant. These enzymes could be the first line of defence in the cellular antioxidant defence pathway, activated due to exposure to abiotic stresses.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Plantas/genética , Plantas/enzimologia , Plantas/genética , Sequência de Aminoácidos , Antioxidantes/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Ascorbato Peroxidases/classificação , Ascorbato Peroxidases/genética , Catalase/classificação , Catalase/genética , Simulação por Computador , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mostardeira/enzimologia , Mostardeira/genética , Oryza/enzimologia , Oryza/genética , Peroxidases/classificação , Peroxidases/genética , Filogenia , Proteínas de Plantas/classificação , Plantas/classificação , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Triticum/enzimologia , Triticum/genéticaRESUMO
Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.
Assuntos
Imunidade/imunologia , Leishmania mexicana/imunologia , Psychodidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Serratia marcescens/imunologia , Sequência de Aminoácidos , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Catalase/classificação , Catalase/genética , Catalase/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade/efeitos dos fármacos , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/imunologia , Insetos Vetores/microbiologia , Insetos Vetores/parasitologia , Leishmania mexicana/fisiologia , Dados de Sequência Molecular , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Psychodidae/enzimologia , Psychodidae/genética , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Serratia marcescens/fisiologia , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologiaRESUMO
Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.
Assuntos
Superóxido Dismutase/isolamento & purificação , Trypanosoma cruzi/enzimologia , Animais , Superóxido Dismutase/classificaçãoRESUMO
Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85 percent, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.