destruction of bacterial spores by solar uv radiation” · “destruction of bacterial spores by...

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1 Workshop on CBRN Defence 22-24 October 2013 Brussels De struction of Bac terial Spores by S olar UV Radiation” Dr. Ralf Möller (representing the DEBACS project team) German Aerospace Center (DLR e.V.) Institute of Aerospace Medicine Radiation Biology Department, Cologne, Germany C ollaborative L inkage G rant (CLG) number: CBP.EAP.CLG.983747 status: completed (2009-2011) Brussels, Belgium, 23 October 2013

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Workshop on CBRN Defence – 22-24 October 2013 – Brussels

“Destruction of Bacterial Spores by Solar UV Radiation”

Dr. Ralf Möller (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

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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

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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

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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, 55°C)

B. cereus (food-poising)

B. anthracis (the bioterrorist)

B. thuringiensis (the exterminator)

Food

Description: background

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UV radiation areas and reactivity

UV radiation Reactivity

- different λ 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

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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

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Direct or indirect radiation damage

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

(i) repair

(ii) detoxification

Description: effects of radiation

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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

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Working flow from the spore exposure to environmental UV

radiation until survival, mutation screening and UV-radiation

damage analyses.

Description: workflow of project

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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

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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

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WP2: UV generated DNA lesions

Spore DNA fragmentation after exposure to terrestrial UV

Outcome and results: DNA damage

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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

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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

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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

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WP3: Mutagenicity of UV radiation

LB medium / LB with 50 µg/ml Rif

• increase UV

• increase RifR

• antibioticR & UVR

= costs of spore inactivation?

Outcome and results: mutation induction

UV-C UV-(A+B)

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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

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CBP.EAP.CLG.983747

• all members of the DEBACS team: Krunoslav Brčić-Kostić, Jose-Luis Sagripanti , Ignacija Vlasic.

• colleagues and collaborators

• technical assistance (Andrea Schröder)

• NATO: Science for Peace and Security Programme

Acknowledgements

Acknowledgements