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  • 1

    Workshop on CBRN Defence 22-24 October 2013 Brussels

    Destruction of Bacterial Spores by Solar UV Radiation

    Dr. Ralf Mller (representing the DEBACS project team)

    German Aerospace Center (DLR e.V.)

    Institute of Aerospace Medicine

    Radiation Biology Department, Cologne, Germany

    Collaborative Linkage Grant (CLG) number: CBP.EAP.CLG.983747

    status: completed (2009-2011)

    Brussels, Belgium, 23 October 2013

  • 2

    Project: Sporicidal effects of UV

    Microbial inactivation via UV radiation: impact for CBRN defence program

    Bacillus subtilis spores biological dosimeter for testing UV radiation sources

    Spore resistance to germicidal and environmental UV

    Overview

    Overview

  • 3

    Project: Sporicidal effects of UV

    - determination of spore resistance to germicidal and environmental UV radiation

    - characterization of the mechanisms allowing spores to survive/resist UV radiation

    - recording of fluence-effect correlation and inactivation rates (decimal reduction value)

    - characterization of UV-induced DNA photoproducts

    - studying the (potential) mutagenic effects of UV radiation

    Objectives

    Objectives

  • 4

    Sky

    Soil Hay Desert Rocks

    Deep surface

    Pathogens Insects

    Where can we find spores of Bacillus spp.?

    B. stratosphericus (above 24 km)

    B. subtilis (hay-Bacillus)

    B. infernus (2700 m below surface)

    B. sonorensis (Sonoran Desert,

    Arizona, USA)

    B. simplex (500 spores/g rock) B. thermoterrestis

    (egypt. soil, 55C)

    B. cereus (food-poising)

    B. anthracis (the bioterrorist)

    B. thuringiensis (the exterminator)

    Food

    Description: background

  • 5

    UV radiation areas and reactivity

    UV radiation Reactivity

    - different area (10-400 nm)

    - environmental (290-400 nm)

    [natural insolation]

    - artificial (254 nm) [industrial / military / clinical application]

    > selective absorption by

    molecules (DNA, RNA,

    proteins)

    > interaction and

    biochemical changes

    > major DNA damage

    (dimers, SSB, DSB)

    Description: background

  • 6

    Portrait of a Bacillus subtilis spore

    1) DNA profile

    A-DNA conformation

    one single genome copy

    toroidal-shaped chromosome

    SASPs binding

    2) Spore interior

    rRNA, ribosomes

    low water content

    Ca2+-DPA complex

    minerals, 3-PGA

    3) Spore exterior

    spore coat and pigments

    crust and exosporium

    Nicholson et al., Microbiol. Mol. Biol. Rev. (2000)

    Description: background

  • 7

    Direct or indirect radiation damage

    Horneck et al., Microbiol. Mol. Biol. Rev. (2010)

    (i) repair

    (ii) detoxification

    Description: effects of radiation

  • 8

    Tools, approaches and methods to

    study UV radiation effects Galactic Cosmic Rays (GCR):

    high energy protons and heavy ions 87 % Protons 12 % -Particles 1 % heavy ions

    UV radiation

    (254 nm)

    Biological model system:

    - Spores of Bacillus subtilis (wild-type, mutant (DPA, SASP formation, increased core water content, pigmentation, coat assembly) and DNA repair deficient-strains (HR, NHEJ, AP, SP lyase, TLS))

    UV-C lamps and sunlight simulators

    Experimental approach:

    Assays:

    Description: objectives

  • 9

    Working flow from the spore exposure to environmental UV

    radiation until survival, mutation screening and UV-radiation

    damage analyses.

    Description: workflow of project

  • 10

    WP1: Response to UV-C radiation (254 nm)

    Galactic Cosmic Rays (GCR):

    high energy protons and heavy ions 87 % Protons 12 % -Particles 1 % heavy ions

    Setlow, JAM (2006), Moeller et al., J. Bacteriol. (2007,08,09,11) Rivas-Castillo et al., Curr. Microbiol (2011), Nicholson et al., MMBR (2000)

    Protection: SASP, core water content, DPA Repair: SP lyase, NHEJ, HR, AP, TLS

    Outcome and results: radiation resistance

  • 11

    WP1: Spore resistance to environmental

    relevant UV radiation

    290-400 nm 320-400 nm

    Protection: SASP, DPA, coat layers, pigm., core water Repair: SP lyase, NHEJ, HR, AP

    Moeller et al. in J. Bacteriol. (2007,09,11)

    Outcome and results: radiation resistance

  • 12

    WP2: UV generated DNA lesions

    Spore DNA fragmentation after exposure to terrestrial UV

    Outcome and results: DNA damage

  • 13

    WP2: DNA photoproducts (UV-C)

    DNA-protein interaction (SASP: small, acid-soluble spore proteins)

    wild-type: 1 molecule SASP

    every 5 bp spore DNA

    SASP mutant:

    1 molecule SASP

    every 20-25 bp spore DNA

    in cooperation with T. Douki and J. Cadet

    Setlow, JAM (2006), Moeller et al., Int. Microbiol. (2007), Lee et al., PNAS (2008)

    same induction rate of total DNA photoproducts

    Outcome and results: DNA damage

  • 14

    WP2: DNA photoproducts (UV-C)

    different spectra of DNA lesions (SP, CTP, 6-4) Setlow, JAM (2006)

    Moeller et al., Int. Microbiol. (2007)

    Outcome and results: DNA damage

  • 15

    WP3: Mutagenicity of UV radiation

    Moeller et al., J. Bacteriol. (2007,08,09,11)

    254-nm UV-C 290-400 UV-(A+B) 320-400 UV-A

    Error prone vs. free DNA repair: AP Protection: SASP

    NalR hot spots: in GyrA: S63F/L, S84L, E88Q

    Outcome and results: mutation induction

  • 16

    WP3: Mutagenicity of UV radiation

    LB medium / LB with 50 g/ml Rif

    increase UV

    increase RifR

    antibioticR & UVR

    = costs of spore inactivation?

    Outcome and results: mutation induction

    UV-C UV-(A+B)

  • 17

    Summary and outlook

    Project-related outcome

    Insights in the UV resistance/inactivation of bacterial spores (CBRN defence)

    Determination of types and nature of DNA lesions / combined treatment

    Occurrence of antibiotic resistant strains (UVR) / general health concern

    Further research focus: suggestions

    Support of studies on combined treatments (heat, chemicals and radiation)

    Funding (start-up projects, teaching, education, workshop, exchanges for young researchers (PhD students, PostDocs)

    Project evaluation: direct scientific and educational successes

    Publications in microbiology journals (3 published, 1 in progress)

    Research exchange => Guest scientist (incl. further work on DNA repair) regional & international cooperation (involvement & networking)

    Stimulation of new aspects: antibiotic resistant strains & resistance

    Project evaluation, impact, ideas for future SPS activities in CBRN defence

  • 18

    CBP.EAP.CLG.983747

    all members of the DEBACS team: Krunoslav Bri-Kosti, Jose-Luis Sagripanti , Ignacija Vlasic.

    colleagues and collaborators

    technical assistance (Andrea Schrder)

    NATO: Science for Peace and Security Programme

    Acknowledgements

    Acknowledgements

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