UV resistance of Bacillus subtilis spores - Involvement of DPA and characterization by photonic microscopy

Ce projet de thèse a eu pour objectif d’étudier la résistance aux UVc des spores de Bacillus subtilis. La première partie de ce projet s’est focalisée sur la molécule d’acide dipicolinique (DPA) qui est connue pour être impliquée dans la résistance des spores aux UV. Cependant des zones d’ombre restaient encore non élucidées. Il est notamment connu que cette molécule forme des photoproduits fluorescents (DPAp), qui étaient encore non identifiés au début de ce projet. Aucune information concernant le rôle de ces DPAp dans la résistance aux UVc n’était alors disponible. Au cours de ce projet un protocole a été établi pour suivre l’apparition des ces photoproduits directement à l’intérieur du protoplaste des spores. Une méthode a également été mise en place pour introduire artificiellement des DPAp dans des spores. Certains de ces DPAp semblent avoir des effets bénéfiques pour les spores, en augmentant leur taux de germination, leur croissance et leur résistance aux UVc. Enfin, il semble que l’hydratation du DPA au moment de l’exposition aux UVc influence la formation des DPAp. La seconde partie de ce projet a quant à elle été dédié à la mise en place d’une exposition équivalente à de l’ UVc directement sous microscope et l’observation directe des dégâts engendrés sur les spores. L’utilisation d’un laser pulsé infrarouge pour fournir une énergie équivalente à celle des UV a cependant révélé que les dégâts engendrés sont différents de ceux trouvés en UV. Etonnement, les spores ne sont que deux fois plus résistantes que les cellules végétatives face à cette exposition. Les premiers résultats montrent que le DPA ne semble ici par avoir de rôle significatif. Finalement, la dernière partie de ce projet a été consacrée à la mise au point d’un marquage des structures de la spore compatible avec des méthodes d’imagerie super résolue PALM/STORM.The aim of this project was to study the UVc resistance of Bacillus subtilis spores. The first part of this project focused on the dipicolinic acid molecule (DPA) which is known to be involved in the UV resistance of spores. However, some points remain unresolved. In particular, it is known that this molecule forms fluorescent photoproducts (DPAp), which were not identified at the beginning of this project. No information concerning the role of these DPAp in UVC resistance was available at that time. During this project, a protocol was established to follow the appearance of these photoproducts directly inside the spore core. A method was also set up to artificially introduce DPAp into the spores. Some of these DPAp seem to have beneficial effects for the spores, by increasing their germination rate, their growth and their resistance to UVc. Finally, it seems that the hydration of DPA at the time of UVc exposure influences the formation of DPAp. The second part of this project was devoted to setting up a UVc equivalent exposure directly under the microscope and directly observing the damage caused on the spores. Using a pulsed infrared laser to provide UV equivalent energy, however, revealed that the damage caused is different from that found in UV. Surprisingly, the spores were only twice as resistant as the vegetative cells to this exposure. The first results show that DPA does not seem to have a significant role here. Finally, the last part of this project was devoted to the development of a labeling of spore structures compatible with PALM/STORM super-resolved imaging methods

Résistance des spores de Bacillus subtilis face aux UV - Implication du DPA et caractérisation par microscopie photonique

The aim of this project was to study the UVc resistance of Bacillus subtilis spores. The first part of this project focused on the dipicolinic acid molecule (DPA) which is known to be involved in the UV resistance of spores. However, some points remain unresolved. In particular, it is known that this molecule forms fluorescent photoproducts (DPAp), which were not identified at the beginning of this project. No information concerning the role of these DPAp in UVC resistance was available at that time. During this project, a protocol was established to follow the appearance of these photoproducts directly inside the spore core. A method was also set up to artificially introduce DPAp into the spores. Some of these DPAp seem to have beneficial effects for the spores, by increasing their germination rate, their growth and their resistance to UVc. Finally, it seems that the hydration of DPA at the time of UVc exposure influences the formation of DPAp. The second part of this project was devoted to setting up a UVc equivalent exposure directly under the microscope and directly observing the damage caused on the spores. Using a pulsed infrared laser to provide UV equivalent energy, however, revealed that the damage caused is different from that found in UV. Surprisingly, the spores were only twice as resistant as the vegetative cells to this exposure. The first results show that DPA does not seem to have a significant role here. Finally, the last part of this project was devoted to the development of a labeling of spore structures compatible with PALM/STORM super-resolved imaging methods.Ce projet de thèse a eu pour objectif d’étudier la résistance aux UVc des spores de Bacillus subtilis. La première partie de ce projet s’est focalisée sur la molécule d’acide dipicolinique (DPA) qui est connue pour être impliquée dans la résistance des spores aux UV. Cependant des zones d’ombre restaient encore non élucidées. Il est notamment connu que cette molécule forme des photoproduits fluorescents (DPAp), qui étaient encore non identifiés au début de ce projet. Aucune information concernant le rôle de ces DPAp dans la résistance aux UVc n’était alors disponible. Au cours de ce projet un protocole a été établi pour suivre l’apparition des ces photoproduits directement à l’intérieur du protoplaste des spores. Une méthode a également été mise en place pour introduire artificiellement des DPAp dans des spores. Certains de ces DPAp semblent avoir des effets bénéfiques pour les spores, en augmentant leur taux de germination, leur croissance et leur résistance aux UVc. Enfin, il semble que l’hydratation du DPA au moment de l’exposition aux UVc influence la formation des DPAp. La seconde partie de ce projet a quant à elle été dédié à la mise en place d’une exposition équivalente à de l’ UVc directement sous microscope et l’observation directe des dégâts engendrés sur les spores. L’utilisation d’un laser pulsé infrarouge pour fournir une énergie équivalente à celle des UV a cependant révélé que les dégâts engendrés sont différents de ceux trouvés en UV. Etonnement, les spores ne sont que deux fois plus résistantes que les cellules végétatives face à cette exposition. Les premiers résultats montrent que le DPA ne semble ici par avoir de rôle significatif. Finalement, la dernière partie de ce projet a été consacrée à la mise au point d’un marquage des structures de la spore compatible avec des méthodes d’imagerie super résolue PALM/STORM

Résistance des spores de Bacillus subtilis face aux UV - Implication du DPA et caractérisation par microscopie photonique

The aim of this project was to study the UVc resistance of Bacillus subtilis spores. The first part of this project focused on the dipicolinic acid molecule (DPA) which is known to be involved in the UV resistance of spores. However, some points remain unresolved. In particular, it is known that this molecule forms fluorescent photoproducts (DPAp), which were not identified at the beginning of this project. No information concerning the role of these DPAp in UVC resistance was available at that time. During this project, a protocol was established to follow the appearance of these photoproducts directly inside the spore core. A method was also set up to artificially introduce DPAp into the spores. Some of these DPAp seem to have beneficial effects for the spores, by increasing their germination rate, their growth and their resistance to UVc. Finally, it seems that the hydration of DPA at the time of UVc exposure influences the formation of DPAp. The second part of this project was devoted to setting up a UVc equivalent exposure directly under the microscope and directly observing the damage caused on the spores. Using a pulsed infrared laser to provide UV equivalent energy, however, revealed that the damage caused is different from that found in UV. Surprisingly, the spores were only twice as resistant as the vegetative cells to this exposure. The first results show that DPA does not seem to have a significant role here. Finally, the last part of this project was devoted to the development of a labeling of spore structures compatible with PALM/STORM super-resolved imaging methods.Ce projet de thèse a eu pour objectif d’étudier la résistance aux UVc des spores de Bacillus subtilis. La première partie de ce projet s’est focalisée sur la molécule d’acide dipicolinique (DPA) qui est connue pour être impliquée dans la résistance des spores aux UV. Cependant des zones d’ombre restaient encore non élucidées. Il est notamment connu que cette molécule forme des photoproduits fluorescents (DPAp), qui étaient encore non identifiés au début de ce projet. Aucune information concernant le rôle de ces DPAp dans la résistance aux UVc n’était alors disponible. Au cours de ce projet un protocole a été établi pour suivre l’apparition des ces photoproduits directement à l’intérieur du protoplaste des spores. Une méthode a également été mise en place pour introduire artificiellement des DPAp dans des spores. Certains de ces DPAp semblent avoir des effets bénéfiques pour les spores, en augmentant leur taux de germination, leur croissance et leur résistance aux UVc. Enfin, il semble que l’hydratation du DPA au moment de l’exposition aux UVc influence la formation des DPAp. La seconde partie de ce projet a quant à elle été dédié à la mise en place d’une exposition équivalente à de l’ UVc directement sous microscope et l’observation directe des dégâts engendrés sur les spores. L’utilisation d’un laser pulsé infrarouge pour fournir une énergie équivalente à celle des UV a cependant révélé que les dégâts engendrés sont différents de ceux trouvés en UV. Etonnement, les spores ne sont que deux fois plus résistantes que les cellules végétatives face à cette exposition. Les premiers résultats montrent que le DPA ne semble ici par avoir de rôle significatif. Finalement, la dernière partie de ce projet a été consacrée à la mise au point d’un marquage des structures de la spore compatible avec des méthodes d’imagerie super résolue PALM/STORM

Effects of pulsed near infrared light (NIR) on Bacillus subtilis spores

International audienceIn this study, we develop a characterization of bacterial spore resistance to NIR pulsed light under modalities traditionally used in multiphoton microscopy. Energy dose and laser power are both key parameters in spore and bacterial cell inactivation. Surprisingly, spores and vegetative cells seem to show a similar sensitivity to pulsed NIR, spores being only 2-fold more resistant than their vegetative counterparts. This work enables us to eliminate certain hypotheses concerning the main driver of spore inactivation processes. Our findings suggest that damage leading to inactivation is mainly caused by photochemical reactions characterized by multiple possible pathways, including DNA damage or oxidation processes

Uptake of UVc induced photoproducts of dipicolinic acid by Bacillus subtilis spores – Effects on the germination and UVc resistance of the spores

International audienceDipicolinic acid (DPA) is a specific molecule of bacterial spores which is essential to their resistance to various stresses such as ultraviolet (UV) exposure and to their germination. DPA has a particular photochemistry that remains imperfectly understood. In particular, due to its ability to absorb UVc radiation, it is likely to form in vitro a wide variety of photoproducts (DPAp) of which only about ten have been recently identified. The photochemical reactions resulting in DPAp, especially those inside the spores, are still poorly understood. Only one of these DPAp, which probably acts as a photosensitizer of DNA upon exposure to UVc, has been identified as having an impact on spores. However, as UVc is required to form DPAp, it is difficult to decouple the overall effect of UVc exposure from the possible effects of DPAp alone. In this study, DPAp were artificially introduced into the spores of the FB122 mutant strain of Bacillus subtilis, one that does not produce DPA. These experiments revealed that some DPAp may play a positive role for the spore. These benefits are visible in an improvement in spore germination rate and kinetics, as well as in an increase in their resistance to UVc exposure

Spectroscopic and microscopic characterization of dipicolinic acid and its salt photoproducts – A UVc effect study on DPA in solution and in bacterial spores

International audienceBacterial spores can cause significant problems such as food poisoning (like neurotoxin or emetic toxin) or serious illnesses (like anthrax or botulism). This dormant form of bacteria, made of several layers of barriers which provide extreme resistance to many abiotic stresses (radiation, temperature, pressure, etc.), are difficult to investigate in situ. To better understand the biological and chemical mechanisms involved and specific to spores resistance, the acquisition of environmental parameters is necessary. For that purpose, our research has been focused on the detection and analysis of a unique spore component, dipicolinic acid (DPA), used as the main in situ metabolite for sporulating bacteria detection. In its native form, DPA is only weakly fluorescent but after Ultraviolet irradiation at the wavelength of 254 nm (UVc), DPA photoproducts (DPAp) exhibit a remarkable fluorescence signal. These photoproducts are rarely identified and part of this study gives new insights offered by mass spectrometry (MS) in the determination of DPA photoproducts. Thanks to DPA assay techniques and fluorescence spectrometry, we highlighted the instability of photoproducts and introduced new assumptions on the effects of UVc on DPA. Studies in spectroscopy and microscopy allowed us to better understand these native probes in bacterial spores and will allow the implementation of a new method for studying the physico-chemical parameters of spore resistance

Influence of hydration on calcium dipicolinate (CaDPA) during UVb and UVc exposure studied via Raman, FTIR and O-PTIR spectroscopy

International audienceCalcium dipicolinate (CaDPA), a major component of bacterial spores, is known to be either a photoprotective or a photosensitizing agent in terms of UV radiation, depending on its hydration state. This molecule is also known to produce numerous photoproducts (DPAp) upon UV exposure, some of which influence spore resistance. The purpose of this study was to better characterize the influence of hydration on the photochemistry of CaDPA using vibrational spectroscopy through three different techniques: Raman, FTIR and O-PTIR microspectroscopy. Synthetic CaDPA was exposed to UVc or UVb in dry conditions or in a water solution. Raman, FTIR and O-PTIR spectra of dry or wet CaDPA were compared before and after irradiation. Results suggest that UVc and UVb exposure mainly affects the bands corresponding to the pyridine ring of CaDPA. However, this effect differs depending on the UV range and the hydration state during UV exposure. Spectra of one DPAp identified in a previous publication as a photosensitizer, named DPAp1, were also acquired by means of the same three spectroscopy techniques. Although DPAp1 comes from a dimerization of two DPA molecules, its spectra appear to be quite different from those of CaDPA. Moreover, this study compares the data provided by O-PTIR, Raman and FTIR spectroscopy via a multiblock analysis. While the information obtained via O-PTIR seems to be closer to that provided by Raman than to that provided by FTIR, the three techniques seem to be quite complementary