Etude de la spore de Bacillus subtilis : caractérisation des structures impliquées dans sa résistance

The bacterial spore is a multilayer microbial form which is extremely resistant to environmental perturbations. This resistance is especially due to its unique structure which is particularly compact and weakly permeable. This work aims to identify and characterize the spore structures involved in these properties. Overall investigation methods, such as NMR and fluorescence anisotropy, have shown that the cortex of Bacillus subtilis spores is modified by temperature for level similar to that of the activation of germination. This will result in changes to the access to the inner membrane. A tool at the spore’s scale, the fluorescence lifetime imaging microscopy (FLIM) in conjunction with the use of a molecular rotor, has been set up. This tool allowed demonstrating that inner membrane of B. subtilis has a very high viscosity, about two times greater than that of the membrane of a vegetative cell. This viscosity is changed by temperature near 65 °C, which corresponds to activation of germination. A stress known to modify the structural integrity of the spore has also been studied: ethanol combined with significant temperature (65 ou 70 °C). This treatment is responsible for inactivation of spores in parallel with their permeabilization. Ethanol especially leads to alteration of the inner membrane for which the viscosity and permeability are changed. These results provide new understanding of mechanisms implicated in spores’ destruction. They allow considering some new applications, for which it is necessary to control structural changing, for example microencapsulation.La spore bactérienne est une forme microbienne multicouche extrêmement résistante aux perturbations environnementales. Cette résistance est notamment liée à sa structure unique qui est particulièrement peu perméable et compacte. Ce travail de thèse a pour but d’identifier et de caractériser les structures sporales impliquées dans ces propriétés. Des méthodes d’investigations globales comme la RMN ou l’anisotropie de fluorescence ont permis de montrer que le cortex des spores de Bacillus subtilis est modifié par la température, pour des valeurs proches de celle de l’activation de la germination. Ceci aura pour conséquence de modifier l’accès à la membrane interne. Un outil d’étude à l’échelle de la spore, l’imagerie en temps de vie de fluorescence (FLIM) couplé à l’utilisation d’un rotor moléculaire, a également été mis au point. Cet outil a permis de mettre en évidence que la membrane interne de B. subtilis possède une très forte viscosité, environ deux fois plus importante que celle de la membrane d’une cellule végétative. Cette viscosité n’est modifiée par la température qu’au-delà de 65 °C, correspondant également à l’activation de la germination. Une perturbation connue pour modifier l’intégrité de la structure de la spore a également été étudiée : l’éthanol couplé à une température importante (65 ou 70°C). Ce traitement est responsable d’une perméabilisation et d’une inactivation des spores. L’éthanol conduit notamment à l’altération de la membrane interne, dont la viscosité et la perméabilité sont modifiées. Ces résultats apportent de nouvelles données pour la compréhension des mécanismes responsables de l’inactivation des spores. Ils permettent d’envisager des applications, pour lesquelles une maitrise des modifications structurales est nécessaire, comme la microencapsulation

Study of Bacillus subtilis spore's : characterication of stuctures implied in its resistance

La spore bactérienne est une forme microbienne multicouche extrêmement résistante aux perturbations environnementales. Cette résistance est notamment liée à sa structure unique qui est particulièrement peu perméable et compacte. Ce travail de thèse a pour but d’identifier et de caractériser les structures sporales impliquées dans ces propriétés. Des méthodes d’investigations globales comme la RMN ou l’anisotropie de fluorescence ont permis de montrer que le cortex des spores de Bacillus subtilis est modifié par la température, pour des valeurs proches de celle de l’activation de la germination. Ceci aura pour conséquence de modifier l’accès à la membrane interne. Un outil d’étude à l’échelle de la spore, l’imagerie en temps de vie de fluorescence (FLIM) couplé à l’utilisation d’un rotor moléculaire, a également été mis au point. Cet outil a permis de mettre en évidence que la membrane interne de B. subtilis possède une très forte viscosité, environ deux fois plus importante que celle de la membrane d’une cellule végétative. Cette viscosité n’est modifiée par la température qu’au-delà de 65 °C, correspondant également à l’activation de la germination. Une perturbation connue pour modifier l’intégrité de la structure de la spore a également été étudiée : l’éthanol couplé à une température importante (65 ou 70°C). Ce traitement est responsable d’une perméabilisation et d’une inactivation des spores. L’éthanol conduit notamment à l’altération de la membrane interne, dont la viscosité et la perméabilité sont modifiées. Ces résultats apportent de nouvelles données pour la compréhension des mécanismes responsables de l’inactivation des spores. Ils permettent d’envisager des applications, pour lesquelles une maitrise des modifications structurales est nécessaire, comme la microencapsulation.The bacterial spore is a multilayer microbial form which is extremely resistant to environmental perturbations. This resistance is especially due to its unique structure which is particularly compact and weakly permeable. This work aims to identify and characterize the spore structures involved in these properties. Overall investigation methods, such as NMR and fluorescence anisotropy, have shown that the cortex of Bacillus subtilis spores is modified by temperature for level similar to that of the activation of germination. This will result in changes to the access to the inner membrane. A tool at the spore’s scale, the fluorescence lifetime imaging microscopy (FLIM) in conjunction with the use of a molecular rotor, has been set up. This tool allowed demonstrating that inner membrane of B. subtilis has a very high viscosity, about two times greater than that of the membrane of a vegetative cell. This viscosity is changed by temperature near 65 °C, which corresponds to activation of germination. A stress known to modify the structural integrity of the spore has also been studied: ethanol combined with significant temperature (65 ou 70 °C). This treatment is responsible for inactivation of spores in parallel with their permeabilization. Ethanol especially leads to alteration of the inner membrane for which the viscosity and permeability are changed. These results provide new understanding of mechanisms implicated in spores’ destruction. They allow considering some new applications, for which it is necessary to control structural changing, for example microencapsulation

Impact of reducing and oxidizing agents on the infectivity of Q beta phage and the overall structure of its capsid

Q beta phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Q beta phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro. Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage.Physiological redox conditions modify disulfide bonds of the capsid of Q beta phage without decreasing infectivity, whereas total reduction disrupts the capsid and chlorine oxidation disrupts especially minor proteins.Physiological redox conditions modify disulfide bonds of the capsid of Q beta phage without decreasing infectivity, whereas total reduction disrupts the capsid and chlorine oxidation disrupts especially minor proteins

Physico-chemical state influences in vitro release profile of curcumin from pectin beads

International audienceCurcumin is a polyphenolic compound with diverse effects interesting to develop health benefit products but its formulation in functional foods or in food supplement is hampered by its poor water solubility and susceptibility to alkaline conditions, light, oxidation and heat. Encapsulation of curcumin could be a mean to overcome these difficulties. In this paper, curcumin was encapsulated by ionotropic gelation method in low methoxyl pectin beads associated with different surfactants: Solutol®, Transcutol® and sodium caseinate. After encapsulation, physico-chemical properties of encapsulated curcumin such as its solubility, physical state, tautomeric forms and encapsulation efficiency as well as encapsulation yield were characterized. In vitro dissolution of curcumin from beads displayed different kinetic profiles according to bead composition due to different matrix network. As Solutol® was a good solvent for curcumin, the drug was present into amorphous form in these beads inducing a rapid release of curcumin in the simulated digestive fluids. In contrast, drug release was slower from sodium caseinate beads since curcumin was not totally dissolved during the manufacturing process. Moreover, the FLIM studies showed that a part of curcumin was encapsulated in caseinate micelles and that 34% of this drug was in keto form which may delay the curcumin release. The Transcutol beads showed also a slow drug release because of the low curcumin solubility and the high density of the matrix

Cartographie de la fluidité des membranes de spores de B. subtilis par microscopie de fluorescence résolue en temps

International audienceL’état de la membrane plasmique des cellules est un élément essentiel pour connaitre la conditionphysiologique des cellules. Sur des microorganismes, cette connaissance permet de mesurer l’impact d’uneperturbation sur la structure cellulaire et sur sa survie ultérieure. La fluidité membranaire résulte tout à lafois de la composition en phospholipides, de la présence et du rôle de certaines molécules comme lesstérols, les protéines transmembranaires mais aussi des conditions thermodynamiques et physico-chimiquesextérieures (P, T, aw). La bactérie Bacillus subtilis est capable dans des conditions défavorables de passerd’un état actif végétatif à l’état de spore, état de dormance accompagné d’un enkystement cellulaire. Danscet état, la structure unique de la spore comporte deux membranes phospholipidiques. La membraneinterne, la plus importante, présente une faible perméabilité sans modifications fondamentales de sacomposition (Griffith and Setlow, 2009). Elle a de plus un rôle essentiel dans l’extrême résistance de la spore,notamment aux attaques chimiques. Afin d’étudier cette transformation ainsi que la résistance de lamembrane de la spore aux différentes perturbations, nous avons développé un nouveau type de marquageafin de suivre la fluidité membranaire dans la cellule et dans la spore. Ce développement a été rendunécessaire car l’imperméabilité de la spore et la présence de deux membranes rendait difficile d’autresapproches. Ce marquage utilise un rotor moléculaire apolaire le Bodipy C12 (Kuimova, 2012). L’utilisation del’imagerie par temps de vie de fluorescence (FLIM) a permis de mesurer directement la microviscosité dumilieu qui environne la sonde ainsi que de différentier le signal venant de chaque membrane (Loison et al.2013). Ce marquage a permis de suivre l’état de la membrane lors de la germination ou lors de perturbationsenvironnementales (éthanol, température). Il devient ainsi possible de suivre l’état membranaire de la sporependant la perturbation. On peut ainsi espérer mieux comprendre comment cette membrane permet à laspore de résister à des conditions extrêmes et comment elle peut être altérée de façon irréversible ou non,pour certaines perturbations

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores:a molecular rotor/FLIM study

AbstractWe utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress

Structural Organizations of Qβ and MS2 Phages Affect Capsid Protein Modifications by Oxidants Hypochlorous Acid and Peroxynitrite

International audiencePathogenic enteric viruses and bacteriophages such as Qβ and MS2 are transmitted through the fecal-oral route. However, oxidants such as peroxynitrite (ONOOH) and hypochlorous acid (HClO) can prevent new infection by inactivating infectious viruses. Their virucidal effect is well recognized, and yet predicting the effects of oxidants on viruses is currently impossible because the detailed mechanisms of viral inactivation remain unclear. Our data show that ONOOH and HClO cross-linked the capsid proteins and RNA genomes of Qβ and MS2 phages. Consistently, the capsids appeared intact by transmission electron microscopy (TEM) even when 99% of the phages were inactivated by oxidation. Moreover, a precise molecular study of the capsid proteins shows that ONOOH and HClO preferentially targeted capsid protein regions containing the oxidant-sensitive amino acid C, Y, or W. Interestingly, the interaction of these amino acids was a crucial parameter defining whether they would be modified by the addition of O, Cl, or NO2 or whether it induced the loss of the protein region detected by mass spectrometry, together suggesting potential sites for cross-link formation. Together, these data show that HClO and ONOOH consistently target oxidant-sensitive amino acids regardless of the structural organization of Qβ and MS2, even though the phenotypes change as a function of the interaction with adjacent proteins/RNA. These data also indicate a potential novel mechanism of viral inactivation in which cross-linking may impair infectivity

Innovative High Gas Pressure Microscopy Chamber Designed for Biological Cell Observation

International audienceAn original high-pressure microscopy chamber has been designed for real-time visualization of biological cell growth during high isostatic (gas or liquid) pressure treatments up to 200 MPa. This new system is highly flexible allowing cell visualization under a wide range of pressure levels as the thickness and the material of the observation window can be easily adapted. Moreover, the design of the observation area allows different microscope objectives to be used as close as possible to the observation window. This chamber can also be temperature controlled. In this study, the resistance and optical properties of this new high-pressure chamber have been tested and characterized. The use of this new chamber was illustrated by a real-time study of the growth of two different yeast strains - Saccharomyces cerevisiae and Candida viswanathii - under high isostatic gas pressure (30 or 20 MPa, respectively). Using image analysis software, we determined the evolution of the area of colonies as a function of time, and thus calculated colony expansion rates

Early exposure to farm dust in an allergic airway inflammation rabbit model: Does it affect bronchial and cough hyperresponsiveness?

International audienceIntroduction Over the past 50 years, the prevalence of allergic respiratory diseases has been increasing. The Hygiene hypothesis explains this progression by the decrease in the bio-diversity of early microbial exposure. This study aims to evaluate the effect of early-life farm exposure on airway hyperresponsiveness and cough hypersensitivity in an allergic airway inflammation rabbit model. Method A specific environment was applied to pregnant rabbits and their offspring until six weeks after birth. Rabbits were housed in a pathogen-free zone for the control group and a calf barn for the farm group. At the end of the specific environmental exposure, both groups were then housed in a conventional zone and then sensitized to ovalbumin. Ten days after sensitization, the rabbit pups received ovalbumin aerosols to provoke airway inflammation. Sensitization to ovalbumin was assessed by specific IgE assay. Cough sensitivity was assessed by mechanical stimulation of the trachea, and bronchial reactivity was assessed by methacholine challenge. The farm environment was characterized by endotoxin measurement. Results A total of 38 rabbit pups were included (18 in the farm group). Endotoxin levels in the farm environment varied from 30 to 1854 EU.m -3 . There was no significant difference in specific IgE values to ovalbumin (p = 0.826) between the two groups. The mechanical threshold to elicit a cough did not differ between the two groups (p = 0.492). There was no difference in the number of cough (p = 0.270) or the intensity of ventilatory responses (p = 0.735). After adjusting for age and weight, there was no difference in respiratory resistance before and after methacholine challenge. Conclusion Early exposure to the calf barn did not affect cough sensitivity or bronchial reactivity in ovalbumin-sensitized rabbits. These results suggest that not all farm environments protect against asthma and atopy. Continuous exposure to several sources of microbial diversity is probably needed