RESUMEN
Cobalt is an essential element for living systems, which, however, make very limited use of this metal, using it mainly in cobalamin-containing enzymes. The reduced use of cobalt compared to other transition metals is generally attributed to the potential toxicity of this element. In this work, we demonstrate that cobalt not only does not have an obvious toxic effect on Salmonella Typhimurium, but that it can efficiently compensate for zinc deficiency in a znuABC deleted strain. In fact, cobalt, but not cobalamin supplementation, rescued all major phenotypic defects of the znuABC strain, including the reduced ability to grow and swim in zinc-deficient media and the high susceptibility to hydrogen peroxide stress. Growth in a cobalt-supplemented defined medium led to the accumulation of large amounts of cobalt both in the wild type and in the znuABC strain. These data suggest that atoms of cobalt may be incorporated in bacterial proteins in place of zinc, ensuring their functionality. In support of this hypothesis we have shown that, in vivo, cobalt can accumulate in ribosomes and replace zinc in a periplasmic Cu,Zn superoxide dismutase (SodCII). Finally, we provide evidence of the ability of cobalt to modulate the intracellular concentration of zinc-regulated proteins (ZnuA, ZinT, and SodCII). Although some observations suggest that in some proteins the replacement of zinc with cobalt can lead to subtle structural changes, the data reported in this study indicate that Salmonella has the ability to use cobalt instead of zinc, without evident harmful effects for cell physiology.
Asunto(s)
Cobalto/metabolismo , Salmonella typhimurium/metabolismo , Zinc/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Humanos , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrolloRESUMEN
Cadmium is a highly toxic metal whose presence in the environment represents a challenge for all forms of life. To improve our knowledge on cadmium toxicity, we have explored Salmonella Typhimurium responses to this metal. We have found that cadmium induces the concomitant expression of the cation efflux pump ZntA and of the high affinity zinc import system ZnuABC. This observation suggests that cadmium accumulation within the cell induces a condition of apparent zinc starvation, possibly due to the ability of this metal to compete with zinc for the metal binding site of proteins. This hypothesis is supported by the finding that strains lacking ZntA or ZnuABC are hyper-susceptible to cadmium and that the cadmium-induced growth defect of a znuABC mutant strain is largely relieved by zinc supplementation. A similar growth defect was observed for a mutant with impaired ability to acquire iron, whereas cadmium does not affect growth of a strain defective in manganese import. Cadmium also influences the expression and activity of the two cytoplasmic superoxide dismutases FeSOD and MnSOD, which are required to control cadmium-mediate oxidative stress. Exposure to cadmium causes a reduction of FeSOD activity in Salmonella wild type and the complete abrogation of its expression in the strain defective in iron import. In contrast, although MnSOD intracellular levels increase in response to cadmium, we observed discrepancies between protein levels and enzymatic activity which are suggestive of incorporation of non-catalytic metals in the active site or to cadmium-mediated inhibition of manganese import. Our results indicate that cadmium interferes with the ability of cells to manage transition metals and highlight the close interconnections between the homeostatic mechanisms regulating the intracellular levels of different metals.
Asunto(s)
Antibacterianos/farmacología , Cadmio/farmacología , Homeostasis , Salmonella typhimurium/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Zinc/metabolismoRESUMEN
Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.
Asunto(s)
Antioxidantes/uso terapéutico , Fibrosis Quística/tratamiento farmacológico , Estrés Oxidativo , Fibrosis Quística/metabolismo , Humanos , Inflamación/tratamiento farmacológicoRESUMEN
Zinc is required for a wide variety of cellular functions and plays a key role in bacterial metabolism and virulence. However, Zn can also be toxic and, therefore, its influx is tightly regulated. The high affinity zinc uptake transporter ZnuABC is the main Zn influx system in Salmonella enterica under conditions of Zn starvation. It has been shown that deletion of the gene encoding for its periplasmic subunit ZnuA significantly affects S. Typhimurium growth rate and virulence, highlighting the importance of this system in the host-pathogen interaction. To gain further insight into the mechanisms involved in Zn influx regulation, we characterized the main alterations in the ionome and proteome of S. Typhimurium wild type and znuA mutant strains grown either under Zn starvation or under Zn-replete conditions. We found significant differences in the element profile and protein expression that were reversed by Zn supplementation. In particular, several of the differentially regulated proteins are predicted to be metal-binding proteins. Interestingly, their over-expression in the znuA mutant strain strictly depends on Zn starvation and correlates with the differences found at the ionome level. In conclusion, our data demonstrate that inhibition of Zn influx has relevant effects either on the bacterial ionome or proteome and shed new light on the role of the ZnuABC system and Zn influx in S. Typhimurium pathogenicity.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteómica , Salmonella enterica/genética , Salmonella enterica/metabolismo , Zinc/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Iones/metabolismo , Fenotipo , Salmonella enterica/efectos de los fármacos , Zinc/farmacologíaRESUMEN
The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.