We recently developed a mathematical model for predicting reactive oxygen species (ROS) concentration and macromolecules oxidation in vivo. We constructed such a model using Escherichia coli as a ...model organism and a set of ordinary differential equations. In order to evaluate the major defences relative roles against hydrogen peroxide (H2 O2), we investigated the relative contributions of the various reactions to the dynamic system and searched for approximate analytical solutions for the explicit expression of changes in H2 O2 internal or external concentrations. Although the key actors in cell defence are enzymes and membrane, a detailed analysis shows that their involvement depends on the H2 O2 concentration level. Actually, the impact of the membrane upon the H2 O2 stress felt by the cell is greater when micromolar H2 O2 is present (9-fold less H2 O2 in the cell than out of the cell) than when millimolar H2 O2 is present (about 2-fold less H2 O2 in the cell than out of the cell). The ratio between maximal external H2 O2 and internal H2 O2 concentration also changes, reducing from 8 to 2 while external H2 O2 concentration increases from micromolar to millimolar. This non-linear behaviour mainly occurs because of the switch in the predominant scavenger from Ahp (Alkyl Hydroperoxide Reductase) to Cat (catalase). The phenomenon changes the internal H2 O2 maximal concentration, which surprisingly does not depend on cell density. The external H2 O2 half-life and the cumulative internal H2 O2 exposure do depend upon cell density. Based on these analyses and in order to introduce a concept of dose response relationship for H2 O2-induced cell death, we developed the concepts of "maximal internal H2 O2 concentration" and "cumulative internal H2 O2 concentration" (e.g. the total amount of H2 O2). We predict that cumulative internal H2 O2 concentration is responsible for the H2 O2-mediated death of bacterial cells.
Here, we show that resistance of Escherichia coli to TiO₂ photocatalysis involves defenses against reactive oxygen species. Results support the idea that TiO₂ photocatalysis generates damage which ...later becomes deleterious during recovery. We found this to be partly due to DNA attack via hydroxyl radicals generated by the Fenton reaction during recovery.
Legionella pneumophila is a waterborne pathogen responsible for Legionnaires' disease, an infection which can lead to potentially fatal pneumonia. After disinfection, L. pneumophila has been ...detected, like many other bacteria, in a "viable but non culturable" state (VBNC). The physiological significance of the VBNC state is unclear and controversial: it could be an adaptive response favoring long-term survival; or the consequence of cellular deterioration which, despite maintenance of certain features of viable cells, leads to death; or an injured state leading to an artificial loss of culturability during the plating procedure. VBNC cells have been found to be resuscitated by contact with amoebae.
We used quantitative microscopic analysis, to investigate this "resuscitation" phenomenon in L. pneumophila in a model involving amending solid plating media with ROS scavengers (pyruvate or glutamate), and co-culture with amoebae. Our results suggest that the restoration observed in the presence of pyruvate and glutamate may be mostly due to the capacity of these molecules to help the injured cells to recover after a stress. We report evidence that this extracellular signal leads to a transition from a not-culturable form to a culturable form of L. pneumophila, providing a technique for recovering virulent and previously uncultivated forms of L. pneumophila.
These new media could be used to reduce the risk of underestimation of counts of virulent of L. pneumophila cells in environmental samples.
Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only ...some proteins are prone to carbonylation.
we used mass spectrometry analysis to identify carbonylated sites in: BSA that had undergone in vitro MCO, and 23 carbonylated proteins in Escherichia coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome.
We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms.
Abstract
The bactericidal effect of photocatalysis with TiO2 is well recognized, although its mode of action is still poorly characterized. It may involve oxidation, as illuminated TiO2 generates ...reactive oxygen species. Here we analyze the bactericidal effect of illuminated TiO2 in NaCl–KCl or sodium phosphate solutions. We found that adsorption of bacteria on the catalyst occurred immediately in NaCl–KCl solution, whereas it was delayed in the sodium phosphate solution. We also show that the rate of adsorption of cells onto TiO2 is positively correlated with its bactericidal effect. Importantly, adsorption was consistently associated with a reduction or loss of bacterial membrane integrity, as revealed by flow cytometry. Our work suggests that adsorption of cells onto aggregated TiO2, followed by loss of membrane integrity, is key to the bactericidal effect of photocatalysis.
Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this ...pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.
Imlay and Linn show that exposure of logarithmically growing Escherichia coli to hydrogen peroxide (H2O2) leads to two kinetically distinguishable modes of cell killing. Mode one killing is ...pronounced near 1 mM concentration of H2O2 and is caused by DNA damage, whereas mode-two killing requires higher concentration (>10 mM). The second mode seems to be essentially due to damage to all macromolecules. This phenomenon has also been observed in Fenton in vitro systems with DNA nicking caused by hydroxyl radical (HO•).
To our knowledge, there is currently no mathematical model for predicting mode one killing in vitro or in vivo after H2O2 exposure.
We propose a simple model, using Escherichia coli as a model organism and a set of ordinary differential equations. Using this model, we show that available iron and cell density, two factors potentially involved in ROS dynamics, play a major role in the prediction of the experimental results obtained by our team and in previous studies. Indeed the presence of the mode one killing is strongly related to those two parameters.
To our knowledge, mode-one death has not previously been explained. Imlay and Linn (Imlay and Linn, 1986) suggested that perhaps the amount of the toxic species was reduced at high concentrations of H2O2 because hydroxyl (or other) radicals might be quenched directly by hydrogen peroxide with the concomitant formation of superoxide anion (a less toxic species). We demonstrate (mathematically and numerically) that free available iron decrease is necessary to explain mode one killing which cannot appear without it and that H2O2 quenching or consumption is not responsible for mode-one death.
We are able to follow ROS concentration (particularly responsible for mode one killing) after exposure to H2O2. This model therefore allows us to understand two major parameters involved in the presence or not of the first killing mode.
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