RESUMO
Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 µM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16â%), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Pseudomonas putida/metabolismo , Telúrio/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Biomassa , Biotransformação , Mutação , Nanoestruturas/química , Nanoestruturas/toxicidade , Proteínas de Transporte de Fosfato/genética , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/crescimento & desenvolvimento , Telúrio/química , Telúrio/toxicidadeRESUMO
The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy.
Assuntos
Antibacterianos/farmacologia , Vias Biossintéticas , Heme/biossíntese , Metaloides/farmacologia , Telúrio/farmacologia , Ácido Aminolevulínico/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Deficiências de Ferro , Testes de Sensibilidade Microbiana , Modelos Biológicos , Mutação/genética , Protoporfirinas/farmacologia , Superóxidos/metabolismo , Telúrio/toxicidadeRESUMO
Mercury salts and tellurite are among the most toxic compounds for microorganisms on Earth. Bacterial mercury resistance is established mainly via mercury reduction by the mer operon system. However, specific mechanisms underlying tellurite resistance are unknown to date. To identify new mechanisms for tellurite detoxification we demonstrate that mercury resistance mechanisms can trigger cross-protection against tellurite to a group of Pseudomonads isolated from the Chilean Antarctic territory. Sequencing of 16S rRNA of four isolated strains resulted in the identification of three Pseudomonads (ATH-5, ATH-41 and ATH-43) and a Psychrobacter (ATH-62) bacteria species. Phylogenetic analysis showed that ATH strains were related to other species previously isolated from cold aquatic and soil environments. Furthermore, the identified merA genes were related to merA sequences belonging to transposons commonly found in isolated bacteria from mercury contaminated sites. Pseudomonas ATH isolates exhibited increased tellurite resistance only in the presence of mercury, especially ATH-43. Determination of the growth curves, minimal inhibitory concentrations and growth inhibition zones showed different tellurite cross-resistance of the ATH strains and suggested a correlation with the presence of a mer operon. On the other hand, reactive oxygen species levels decreased while the thiol content increased when the isolates were grown in the presence of both toxicants. Finally, qPCR determinations of merA, merC and rpoS transcripts from ATH-43 showed a synergic expression pattern upon combined tellurite and mercury treatments. Altogether, the results suggest that mercury could trigger a cell response that confers mercury and tellurite resistance, and that the underlying mechanism participates in protection against oxidative damage.
Assuntos
Mercúrio/toxicidade , Pseudomonas/efeitos dos fármacos , Pseudomonas/isolamento & purificação , Telúrio/toxicidade , Regiões Antárticas , Chile , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Geografia , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Compostos de Sulfidrila/metabolismoRESUMO
Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQS 3D7 and CQR W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC50 values were 3.7±1.0µM, 1.1±0.2µM and 0.2±0.01µM, respectively. Using an assay performed in the presence of the ER Ca(2+)-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.
Assuntos
Antimaláricos/farmacologia , Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Compostos Organometálicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Telúrio/farmacologia , Antimaláricos/toxicidade , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/toxicidade , Descoberta de Drogas , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/parasitologia , Humanos , Concentração Inibidora 50 , Malária Falciparum/tratamento farmacológico , Compostos Organometálicos/toxicidade , Telúrio/toxicidadeRESUMO
Exposure to the tellurium oxyanion tellurite (TeO3(2-)) results in the establishment of an oxidative stress status in most microorganisms. Usually, bacteria growing in the presence of the toxicant turn black because of the reduction of tellurite (Te(4+)) to the less-toxic elemental tellurium (Te(0)). In vitro, at least part of tellurite reduction occurs enzymatically in a nicotinamide dinucleotide-dependent reaction. In this work, we show that TeO3(2-) reduction by crude extracts of Escherichia coli overexpressing the zwf gene (encoding glucose-6-phosphate dehydrogenase) takes place preferentially in the presence of NADPH instead of NADH. The enzyme responsible for toxicant reduction was identified as 6-phosphogluconate dehydrogenase (Gnd). The gnd gene showed a subtle induction at short times after toxicant exposure while strains lacking gnd were more susceptible to the toxicant. These results suggest that both NADPH-generating enzymes from the pentose phosphate shunt may be involved in tellurite detoxification and resistance in E. coli.
Assuntos
Farmacorresistência Bacteriana , Escherichia coli/enzimologia , Escherichia coli/metabolismo , NADP/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Telúrio/metabolismo , Escherichia coli/efeitos dos fármacos , Inativação Metabólica , Oxirredução , Telúrio/toxicidadeRESUMO
Escherichia coli exposed to tellurite shows augmented membrane lipid peroxidation and ROS content. Also, reduced thiols, protein carbonylation, [Fe-S] center dismantling, and accumulation of key metabolites occur in these bacteria. In spite of this, not much is known about tellurite effects on the E. coli electron transport chain (ETC). In this work, tellurite-mediated damage to the E. coli ETC's NADH dehydrogenases and terminal oxidases was assessed. Mutant lacking ETC components showed delayed growth, decreased oxygen consumption and increased ROS in the presence of the toxicant. Membranes from tellurite-exposed E. coli exhibited decreased oxygen consumption and dNADH/NADH dehydrogenase activity, showing an impairment of NDH-I but not of NDH-II activity. Regarding terminal oxidases, only the bo oxidase complex was affected by tellurite. When assaying NDH-I and NDH-II activity in the presence of superoxide, the NDH-I complex was preferentially damaged. The activity was partly restored in the presence of reducing agents, sulfide and Fe(2+) under anaerobic conditions, suggesting that damage affects NDH-I [4Fe-4S] centers. Finally, augmented membrane protein oxidation along with reduced oxidase activity was observed in the presence of the toxicant. Also, the increased expression of genes encoding alternative terminal oxidases probably reflects a cell's change towards anaerobic respiration when facing tellurite.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , NADH Desidrogenase/metabolismo , Oxirredutases/metabolismo , Telúrio/toxicidade , Aerobiose/efeitos dos fármacos , Anaerobiose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Isoenzimas/genética , Isoenzimas/metabolismo , NADH Desidrogenase/genética , Oxirredução/efeitos dos fármacos , Oxirredutases/genética , Consumo de Oxigênio/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
Tellurite (TeO3(2-)) is harmful for most microorganisms, especially Gram-negative bacteria. Even though tellurite toxicity involves a number of individual aspects, including oxidative stress, malfunctioning of metabolic enzymes and a drop in the reduced thiol pool, among others, the general mechanism of toxicity is rather complex and not completely understood to date. This work focused on DNA microarray analysis to evaluate the Escherichia coli global transcriptomic response when exposed to the toxicant. Confirming previous results, the induction of the oxidative stress response regulator soxS was observed. Upregulation of a number of genes involved in the global stress response, protein folding, redox processes and cell wall organization was also detected. In addition, downregulation of aerobic respiration-related genes suggested a metabolic switch to anaerobic respiration. The expression results were validated through oxygen consumption experiments, which corroborated that tellurite-exposed cells effectively consume oxygen at lower rates than untreated controls.
Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Telúrio/toxicidade , Anaerobiose , Escherichia coli/genética , Análise em Microsséries , Oxigênio/metabolismoRESUMO
The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K substitutions were individually transformed into Escherichia coli Δlpd. Cells expressing the modified genes exhibited decreased pyruvate dehydrogenase, dihydrolipoamide dehydrogenase and TR activity regarding that observed with the wild type A. caviae lpdA gene. In addition, cells expressing the altered lpdA genes showed increased oxidative stress levels and tellurite sensitivity than those carrying the wild type counterpart. The involvement of Cys residues in LpdA's TR activity was analyzed using specific inhibitors that interact with catalytic cysteines and/or disulfide bridges such as aurothiomalate, zinc or nickel. TR activity of purified LpdA was drastically affected by these compounds. Since LpdA belongs to the flavoprotein family, the involvement of the FAD/NAD(P)(+)-binding domain in TR activity was determined. FAD removal from purified LpdA results in loss of TR activity, which was restored with exogenously added FAD. Substitutions in E354, involved in FAD/NADH binding, resulted in low TR activity because of flavin loss. Finally, changing H322 (involved in NAD(+)/NADH binding) by tyrosine also resulted in altered TR activity.
Assuntos
Aeromonas caviae/efeitos dos fármacos , Di-Hidrolipoamida Desidrogenase/metabolismo , Telúrio/química , Di-Hidrolipoamida Desidrogenase/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutagênese Sítio-Dirigida , Oxirredução , Telúrio/toxicidadeRESUMO
Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH), which resulted in α-ketoglutarate (α-KG) accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.
Assuntos
Escherichia coli/efeitos dos fármacos , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxirredutases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Escherichia coli/enzimologia , Complexo Cetoglutarato Desidrogenase/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Telúrio/toxicidadeRESUMO
In this study we have examined the in vivo toxic effects of various organochalcogens on hepatic, renal, glycemic and lipid profile. Diorganotellurium dichloride phosphonate (C1) at all tested doses did not modify serum alanine aminotransferase (ALT) activity in mice. While, 2-butyltellurium furan (C2) and dinaphthalene ditelluride (C3) at a dose of 0.75 and 0.125 mmol/kg caused an increase in aspartate aminotransferase (AST) and ALT activities. Our data showed that C1 caused an increase in urea content at different doses while treatment with C2 and C3 did not modify urea content. Treatment with C2 caused a significant alteration in serum glucose and fructosamine levels which explains the possible toxicity of these compounds. No significant changes were observed for cholesterol and triglycerides levels. These results suggest that organochalcogen compounds presented liver and renal toxicity and also altered glycemic profile which may leads to various clinical complications.