Projet ANR-07-BLAN-0218 – DynaBio : Dynamic imaging of the intercellular space in bacterial biofilms using original nanoprobes and biosensors
Programme : Blanc (BLANC) 2007
Année de financement : 2007
Coordinateur du projet : (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE CENTRE-EST)
Partenaires : Centre national de la recherche scientifique; Centre national de la recherche scientifique; Centre national de la recherche scientifique
Montant : 350000.0 euros
Date de début : 2007
Durée : 48 mois
Résumé : The objective of our project is to study the structure, the reactivity and the variability of the polymeric extracellular matrix, which fills up the intercellular space between bacterial cells composing a biofilm. Biofilms, which consist of an organised population of micro-organisms (e.g. bacteria, yeasts), can be observed adhering to living tissues and to mineral or organic surfaces in any natural or man-made system by means of a highly hydrated, visco-elastic matrix made up of organic polymeric substances (EPS). This intercellular matrix, along with the bacterial surfaces, constitutes the true interface between the cells and their environment. Convergent evidences suggest a permanent re-organization of the matrix as an adaptative response of the microbial community toward changing environment. In response to external changes, bacteria may consume and/or produce a variety of organic exo-heteropolymers (EPS) with different physico-chemical properties, which may act as a defensive barrier against aggressive environmental parameters . In this project we have selected di-oxygen (O2) as an effective and representative stressor. We postulate that the oxidative stress caused by elevated O2 concentration will determine the production of even subtly different polymers/molecules which may cause a major re-organization of the matrix properties, e.g. modifications of the hydrophilic/hydrophobic balance, redox status of the intercellular space, architecture of the biofilm. We wish to characterize such environmental driven changes. We will definitively privilege the development of methods allowing non invasive in situ observations of such variations of the matrix (nanoprobes) as opposed to more classical but invasive methods such as extraction of polymers or use of microelectrodes that are poorly resolutive (at the 3D imaging level) and that do not allow dynamic observation of biofilms overtime. In addition we will define the impact of such matrix modifications on the flux of O2 to the cells by using engineered biosensors. Imaging at micro- and nanoscale the physical properties and the reactivity of the EPS matrix, as well as the distribution of O2-perceiving bacteria in stressed and non-stressed biofilms represent the central challenge of our project. -  In this context, and considering the synergistic competence of the three partners, we propose an original in situ study of the structure, reactivity and variability of the intercellular space in biofilms of the Shewanella oneidensis (a model bacteria species which is currently studied in the partner 1 laboratory and characterized regarding its surface properties, and also used as model in other laboratories for biofilm studies) by means of physico-chemical and biological characterization. The biofilms will be submitted to an oxidative stress generated by O2 as this key parameter affecting cell physiology is easily controlled in both laboratory and industrial processes. Indeed, O2 is the cornerstone of numerous metabolisms. It is both a substrate for aerobic respiration and a toxic compound initiating an oxidative stress, to which, or against which, bacterial cells have to respond to either by using it or by preventing its deleterious effects. Our central hypothesis is that, under an oxidative stress, the bacterial community embedded in the biofilm responds by re-organizing the intercellular space in order to protect the cells from O2 and reactive oxygen species (ROS): we assume that the cells will change the properties of their EPS matrix, making them more hydrophobic, which could result in O2 diffusion limitation. Also, anti-oxidant species, such as GSH, for example, could be exported in the intercellular space. To our knowledge, cell responses (mainly intracellular changes) have been well documented, but the re-organization of the extracellular compartment (EPS matrix properties) within a biofilm has never been clearly demonstrated. -  	With this project, we propose to study the spatial