Etudes biochimiques et génétiques de la réponse adaptive de molusques face aux contaminations métalliques et au stress oxydant

Ce travail consiste à étudier au niveau biochimique et génétique la réponse adaptative des mollusques face aux contaminations métalliques et au stress oxydant qui en découle : il s'agit de mettre en évidence des gènes impliqués dans la résistance métallique, d'étudier leur expression et leur régulation, et de déterminer le rôle des protéines correspondantes. Essentiellement deux types de stratégies de défense au niveau cellulaire sont étudiées : l'une consiste à séquestrer le métal dans la cellule grâce à des métallothionéines (MT), l'autre à l'expulser grâce à des protéines transmembranaires, telles que les protéines MDR (multidrug resistance). Outre les bivalves, des systèmes bactériens ont été exploités pour évaluer l'importance de ces protéines dans la défense contre une contamination métallique. Des bactéries exprimant des MTs ont été utilisés pour analyser le rôle dans la séquestration des métaux mais aussi les propriétés oxydoréductrices de ces protéines. Il fut révélé un rôle pro-oxydant des MTs dans certaines conditions. Des vésicules inversées préparées à partir d'un de ces systèmes bactériens ont permis de mettre en évidence une expulsion du cadmium dépendante de l'ATP par une protéine MDR.This work consisted in making biochemical and genetic studies of molluscs adaptive response to metallic contamination and to oxidative stress attributable to such contamination. The objectives were to identify genes involved in metal resistance, to study their expression and regulation, and to determine the role of corresponding proteins in metallic and/or oxidative stress resistance mechanism. Two kinds of resistance strategies where mainly studied: metal sequestration by metallothioneins (MTs) and active extrusion of metals by MDR (multidrug resistance) proteins which are present in the cell membrane. Besides bivalve molluscs, bacteria were used as a cell reactor in order to evaluate the importance of such proteins in metal resistance. Bacteria expressing MTs were used to study the metal sequestration capability and the antioxidant properties of MTs. A pro-oxidant role of MTs has been observed in certain conditions. Inside-out membrane vesicles isolated from bacteria overexpressing an MDR protein gene were used to show the ability of these proteins to transport cadmium in an ATP-dependent way

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 mu M verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent Cd-109(2+) uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metal-extrusion abilities

Danio rerio ABC transporter genes abcb3 and abcb7 play a protecting role against metal contamination

International audienceATP‐binding cassette (ABC) proteins are efflux transporters and some of them are involved in xenobiotic detoxification. The involvement of four zebrafish ABC transporters in cadmium, zinc and mercury detoxification was characterized in a metal hypersensitive mutant of Escherichia coli. The E. coli tolC mutant expressing ABCB3 or ABCB7 transporters exhibited higher survival ratios and lower metal accumulation under a metal exposure condition than the controls. For instance, in the presence of 8 and 10 μM of HgCl2, the survival ratios of bacteria expressing ABCB3 were four and six‐times higher than the control whereas the mercury concentrations were 2.5 and 2‐times lower than in the control. This work provides new data on the function of zebrafish ABCB3 and ABCB7 transporters and highlights their significance in metal detoxificatio

Proteomic and Physiological Responses of Kineococcus radiotolerans to Copper

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0–1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R2 = 0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration

cDNA cloning and gene expression of ribosomal S9 protein gene in the mollusk Corbicula fluminea: A new potential biomarker of metal contamination up-regulated by cadmium and repressed by zinc

International audienceBiological indicators can be used to assess polluted sites, but their success depends on their specificity. The aim of the present study was to identify a specific cadmium biomarker of the freshwater bivalve Corbicula fluminea. Differential display technique was used to identify new molecular indicators for cadmium exposure. Gene expression of C. fluminea transplanted into a cadmium- and zinc-contaminated river was compared to gene expression of bivalves from a reference site. One differentially expressed band was identified, and the entire cDNA sequence coding for the ribosomal protein S9 (rpS9) was cloned. The rpS9 gene expression was studied by real-time reverse transcription-polymerase chain reaction. After controlled cadmium (15 microg/L) and/or zinc (1 mg/L) laboratory contaminations of the bivalves, this gene was shown to be up-regulated by cadmium and down-regulated by zinc. A 48-d transplantation of animals into a polymetallic field contamination in the Lot river basin (Midi-Pyrénées region, France) showed that rpS9 gene regulation was correlated with the fluctuating cadmium and zinc water contents. To our knowledge, the existence of a gene that is up-regulated by cadmium and repressed by zinc is demonstrated here for the first time

Cytochrome c Oxydase Subunit I Gene is Up-regulated by Cadmium in Freshwater and Marine Bivalves

International audienceInhibition of the mitochondrial electron transfer chain and induction of reactive oxygen species (ROS) production are one of the roots of cadmium (Cd) toxicity. To appreciate the impact of Cd on mitochondria, we focused on the expression of CoxI gene which encodes the subunit I of the Cytochrome c oxidase (complex IV of the respiratory chain). CoxI gene expression was studied by real-time quantitative PCR in three species: two freshwater bivalves (Corbicula fluminea and Dreissena polymorpha) and one marine bivalve (diploid or triploid Crassostrea gigas). Bivalves were exposed for 10 or 14 days to 0.13 μM Cd$^2$$^+$ and 15.3 μM Zn$^2$$^+$ in controlled laboratory conditions. We demonstrate that in the three mollusk species CoxI gene was up-regulated by Cd. Zinc (Zn), which is known to have antioxidant properties, had no effect on CoxI gene expression. In the presence of Cd and Zn, CoxI gene inducibility was lower than after a single Cd exposure, in each species; result that could not be fully explained by a decreased Cd accumulation. CoxI gene induction by Cd was 4.8-fold higher in triploid oysters than in diploid ones, indicating a possible influence of triploidy on animal responses to Cd contamination

Heterologously Expressed Bacterial and Human Multidrug Resistance Proteins Confer Cadmium Resistance to Escherichia coli

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 µM verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent 109Cd2+ uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metalextrusion abilities.

Role of metallothioneins in superoxide radical generation during copper redox cycling: Defining the fundamental function of metallothioneins

International audienceIn order to demonstrate the in vivo antioxidant properties of metallothioneins (MTs), the bacteria Escherichia coli was used as a cell reactor in which we compared the metal binding and antioxidative functions of MTs from different species, with different structures and polypeptide lengths. No protective effects of cytoplasmic MTs from cadmium (Cd) or zinc (Zn) contamination were observed in a wild-type E. coli strain, although these MTs can efficiently bind both Cd and Zn. To test their antioxidant properties, MTs were expressed within the cytoplasm of a sodA sodB deficient mutated strain (QC1726). However, a paradoxical MT toxicity was found when this strain was contaminated with Cd and Zn, suggesting that in a wild-type strain, superoxide dismutase counteracts MT toxicity. The most toxic MT was the one with the strongest Cd and Zn binding capacities. This toxic effect was linked to the generation of superoxide radicals, since a Cd-contaminated QC1726 strain expressing oyster MT isoforms produced 75-85% more O(2)*(-) than the control QC1726 strain. Conversely, under anaerobiosis or in the presence of a copper chelator, MTs protected QC1726 strain from Cd and Zn contamination. A model is proposed to explain the observed MT toxicity