[The oyster Crassostrea gigas, a new model against cancer]. / Crassostrea gigas, une huître au service de la recherche sur le cancer.

The Warburg effect is one of the hallmarks of cancer cells in humans. It is a true metabolic reprogramming to aerobic glycolysis, allowing cancer cells to meet their particular energy needs for growth, proliferation, and resistance to apoptosis, depending on the microenvironment they encounter within the tumor. We have recently discovered that the Crassostrea gigas oyster can naturally reprogram its metabolism to the Warburg effect. Thus, the oyster becomes a new invertebrate model useful for cancer research. Due to its lifestyle, the oyster C. gigas has special abilities to adapt its metabolism to the extreme changes in the environment in which it is located. The oyster C. gigas is therefore a model of interest to study how the environment can control the Warburg effect under conditions that could not be explored in vertebrate model species.

Energy and antioxidant responses of pacific oyster exposed to trace levels of pesticides.

Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 µg/L), isoproturon (0.1 and 1 µg/L), or both in a mixture (0.2 and 0.1 µg/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase α (AMPKα), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was up-regulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.

Proteome phenotyping of ΔrelA mutants in Enterococcus faecalis V583.

The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583. A 2-dimensional electrophoresis proteomic analysis of 2 relA mutants, i.e., ΔrelA carrying a complete deletion of the relA gene, and ΔrelAsp that is deleted from only its 3' extremity, showed that 31 proteins were deregulated in 1 or both of these mutants. Mass spectrometry identification of these proteins showed that 10 are related to translation, including 5 ribosomal proteins, 3 proteins involved in translation elongation, and 2 proteins in tRNA synthesis; 14 proteins are involved in diverse metabolisms and biosynthesis (8 in sugar and energy metabolisms, 2 in fatty acid biosynthesis, 2 in amino acid biosynthesis, and 2 in nucleotide metabolism). Five proteins were relevant to the adaptation to different environmental stresses, i.e., SodA and a Dps family protein, 2 cold-shock domain proteins, and Ef1744, which is a general stress protein that plays an important role in the response to ethanol stress. The potential role of these proteins in the development of stress phenotypes associated with these mutations is discussed.

Variation patterns in individual fish responses to chemical stress among estuaries, seasons and genders: the case of the European flounder (Platichthys flesus) in the Bay of Biscay.

The objective was to describe and model variation patterns in individual fish responses to contaminants among estuaries, season and gender. Two hundred twenty-seven adult European flounders were collected in two seasons (winter and summer) in four estuaries along the Bay of Biscay (South West France), focusing on a pristine system (the Ster), vs. three estuaries displaying contrasted levels of contaminants (the Vilaine, Loire and Gironde). Twenty-three variables were measured by fish, considering the load of contaminants (liver metals, liver and muscle persistent organic pollutants, muscle polycyclic aromatic hydrocarbons); the gene expression (Cyt C oxydase, ATPase, BHMT, Cyt P450 1A1, ferritin); the blood genotoxicity (Comet test); and liver histology (foci of cellular alteration-tumour, steatosis, inflammation, abnormal glycogen storage). Canonical redundancy analysis (RDA) was used to model these variables using gender, season and estuary of origin as explanatory variables. The results underlined the homogeneity of fish responses within the pristine site (Ster) and more important seasonal variability within the three contaminated systems. The complete model RDA was significant and explained 35 % of total variance. Estuary and season respectively explained 30 and 5 % of the total independent variation components, whilst gender was not a significant factor. The first axis of the RDA explains nearly 27 % of the total variance and mostly represents a gradient of contamination. The links between the load of contaminants, the expression of several genes and the biomarkers were analysed considering different levels of chemical stress and a possible multi-stress, particularly in the Vilaine estuary.

The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583.

Guanosine penta- and tetraphosphate [(p)ppGpp] are two unusual nucleotides implied in the bacterial stringent response. In many pathogenic bacteria, mutants unable to synthesize these molecules lose their virulence. In Gram-positive bacteria such as Enterococcus faecalis, the synthesis and degradation of (p)ppGpp mainly depend on the activity of a bifunctional enzyme, encoded by the relA gene. By analysing DeltarelA and DeltarelQ (which encodes a protein harbouring a ppGpp synthetase activity) deletion mutants, we showed that RelA is by far the main system leading to (p)ppGpp production under our experimental conditions, and during the development of a stringent response induced by mupirocin. We also constructed a mutant (DeltarelAsp) in which a small part of the relA gene (about 0.7 kbp) encoding the carboxy-terminal domain of the RelA protein was deleted. Both relA mutants were more resistant than the wild-type strain to 0.3 % bile salts, 25 % ethanol and acid (pH 2.3) challenges. Interestingly, the DeltarelAsp mutant grew better than the two other strains in the presence of 1 mM H(2)O(2), but did not display increased tolerance when subjected to lethal doses of H(2)O(2) (45 mM). By contrast, the DeltarelA mutant was highly sensitive to 45 mM H(2)O(2) and displayed reduced growth in a medium containing 1 M NaCl. The two mutants also displayed contrasting virulence phenotypes towards larvae of the Greater Wax Moth infection model Galleria mellonella. Indeed, although the DeltarelA mutant did not display any phenotype, the DeltarelAsp mutant was more virulent than the wild-type strain. This virulent phenotype should stem from its increased ability to proliferate under oxidative environments.

Transcriptional analysis of the cyclopropane fatty acid synthase gene of Lactococcus lactis MG1363 at low pH.

Cyclopropane fatty acid synthase (cfa) catalyses the transfer of a methyl group from S-adenosylmethionine (SAM) to unsaturated fatty acids. Northern blot experiments demonstrated that the Lactococcus lactis MG1363 cfa gene is mainly expressed as a bicistronic transcript together with metK, the gene encoding SAM synthetase, and is highly induced by acidity. The cfa promoter was characterized by 5'-RACE PCR, and fused to beta-galactosidase by cloning into the pAK80 plasmid. This transcriptional fusion was highly induced by acidity (23-fold at pH 5) as well as during entry into the stationary phase (8-fold) in L. lactis. Interestingly, the cfa promoter expression is repressed in a L. lactis relA* mutant which accumulates (p)ppGpp, whereas its induction by acidity appeared independent of (p)ppGpp in L. lactis and in Escherichia coli.

Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis.

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.

The osmoprotectant glycine betaine inhibits salt-induced cross-tolerance towards lethal treatment in Enterococcus faecalis.

The response of Enterococcus faecalis ATCC 19433 to salt stress has been characterized previously in complex media. In this report, it has been demonstrated that this bacterium actively accumulates the osmoprotectant glycine betaine (GB) from salt-enriched complex medium BHI. To further understand the specific effects of GB and other osmoprotective compounds in salt adaptation and salt-induced cross-tolerance to lethal challenges, a chemically defined medium lacking putative osmoprotectants was used. In this medium, bacterial growth was significantly reduced by increasing concentrations of NaCl. At 0.75 M NaCl, 90% inhibition of the growth rate was observed; GB and its structural analogues restored growth to the non-salt-stressed level. In contrast, proline, pipecolate and ectoine did not allow growth recovery of stressed cells. Kinetic studies showed that the uptake of betaines shows strong structural specificity and occurs through a salt-stress-inducible high-affinity porter [Km = 3.3 microM; Vmax = 130 nmol min(-1) (mg protein)(-1); the uptake activity increased 400-fold in the presence of 0.5 M NaCl]. Moreover, GB and its analogues were accumulated as non-metabolizable cytosolic osmolytes and reached intracellular levels ranging from 1-3 to 1.5 micromol (mg protein)(-1). In contrast to the beneficial effect of GB on the growth of salt-stressed cultures of E. faecalis, its accumulation inhibits the salt-induced cross-tolerance to a heterologous lethal challenge. Indeed, pretreatment of bacterial cells with 0.5 M NaCl induced resistance to 0.3% bile salts (survival of adapted cells increased by a factor of 6800). The presence of GB in the adaptation medium reduced the acquisition of bile salts resistance 680-fold. The synthesis of 11 of the 13 proteins induced during salt adaptation was significantly reduced in the presence of GB. These results raise questions about the actual beneficial effect of GB in natural environments where bacteria are often subjected to various stresses.