Transcript Deinococcus radiodurans Presentation

Deinococcus Launches Aboard Endeavor’s Last Flight
The four life forms flown in
Shuttle LIFE were Tardigrades;
the bacteria Deinococcus
radiodurans and Bacillus
subtilis; and the archaeon
Pyrococcus furiosus. A
passenger manifest explained
what characteristics of the
different microorganisms such as resistance to radiation,
and extreme hardiness - made
them good choices for space
travel.
May, 2011
MCB Seminar
April 25, 2013
3:30 pm - 4:30 pm
Lecture Room C
USUHS
RadiationResistant
Deinococcus
radiodurans
Deinococcus Mn2+ Complexes: New Frontiers
Michael J. Daly
Uniformed Services University of the Health Sciences (USUHS)
Bethesda, MD 20814, USA
Email: [email protected]
Deinococcus History
The Paradox
Mn2+ Antioxidants
Applications
Protein
Protection
Ongoing Fukushima Disaster Reminds the World that
Research on Prevention of Acute and Chronic Radiation
Effects is Critical
“Perhaps if we knew why Deinococcus cells are so resistant to radiation, we could find ways to protect
people from atomic radiation”. - A new perspective on radiation resistance. Nature
March 2011: Reactors # 1, 3, 4
Catastrophic
Fukushima
Nuclear
Accident
Leaking Cold War Radioactive Waste
April 8, 2013:
Level 7
Another major radioactive
tank leak discovered
March 10, 2013:
Major radioactive
tank leak discovered
Nuclear Threats
Burns from radiation
devices known since
Thomas Edison’s public
displays of x-rays in 1896
March 26, 2013:
North Korea aims nuclear missile at US
Extreme Ionizing Radiation Resistance
Since 1956, 42 distinct species of Deinococcus reported
Order:
Deinococcales
Genus:
Deinococcus
Species:
D. radiodurans
(Oregon, USA, 1956)
Antarctica
DeinococcusThermus
Nottingham
N. Atlantic
Phylum:
Portugal
D. proteolyticus
Peru
D. radiophilus
Kingdom Bacteria
:
D. geothermalis
60 Gy/hour
D. radiopugnans
137Cs
D. roseus
Japan
D. grandis
3 days, 25oC, 60 Gy/hour on nutrient agar
0.5 mm
http://www.usuhs.mil/pat/deinococcus/index_20.htm
Phylogenetic distribution of radiation resistant organisms. The existence of so many
unrelated radioresistant species suggests that the molecular mechanisms that protect
against ionizing radiation-induced damage evolved independently in these organisms.
Evolving high-level radiation resistance is not so difficult,
as demonstrated in the lab with various bacteria including E. coli
The Model
ROS Production under Aqueous Conditions
by Ionizing Radiation (x-Rays & g-Rays)
Haber-Weiss
Reaction
complexes
1960-2004: As radiation was deemed to damage cellular macromolecules indiscriminately,
and as genes exist at far lower abundance in cells than their products, genes assumed the role
of the most important targets – early on, the DNA double strand break (DSB) was identified
as the critical lesion. And, DNA DSB repair mutants seemed to confirm this – all such
mutants were highly radiation-sensitive.
DNA Repair (Amst). 2012 Jan 2;11(1):12-21 :
DSB Yields for g-Rays
0.2 DSB/Gy/Mbp
0.0005 DSB/Gy/Mbp
0.05 DSB/Gy/Mbp
0.005 DSB/Gy/Mbp
In all cell types tested so far – mammalian cells, simple eukaryotes, archaea, bacteria –
DSB lesion yields for ionizing radiation are essentially the same: ~0.005 DSB/Gy/Mbp
Survival Curves and DSBs
100%
Survival
Representative
Insects
Rotifers
C. elegans
Fungi
Halobacteria
Cyanobacteria
Deinococci
Amoebae
You and
most
of Life
10%
g-Rays
UVC
Desiccation
0
1
0
0
72 DSBs
<1 DSB
5 DSBs
400 DSBs
120 DSBs
10
100
1000
10000
0.1
1
10
100
1000
0.1
1
10
100
1000
g-rays
Dose/Gy
Dose/J/m2
Dose/days
So, the central question became: What is the molecular
basis of extremely efficient DSB repair in Deinococcus?
The New York Times (1999) The D. radiodurans genome does not appear to encode an
unusual set of DNA repair genes that is distinct from those in radiation-sensitive bacteria.
The Paradox
For a given dose of g-radiation, the level of DNA damage in
D. radiodurans compared to all other organisms is very similar
~0.004 DSB/Gy/Haploid Genome
D. radiodurans
17,500 Gy
D. radiodurans
Post-17,500 Gy
24 hours later
20 kb
In 2004, we reported that Mn accumulation closely linked to
Deinococcus radiation resistance. But, Mn didn’t protect DNA
So, What is Mn protecting?
● Deinococcus hoards Mn2+ (0.25-1 mM)
in cytoplasm, but Fe out.
● As resistance in different bacteria
went up, so did their [Mn]
Mn2+
Fe
Yet, for a given dose of g-radiation, the level of protein damage
in Deinococcus compared to other bacteria is very different
Fe
Mn
The Bacteria
Exposed to
Same Dose
Same Protein
Purification
Procedure
Protein
Carbonyl
The Paradox
Resistance
Protein
Oxidation
Assay
A founding concept of radiobiology that deals with X-rays + g-rays
is that radiation indiscriminately damages cellular macromolecules.
Whereas DNA lesion-yields in cells exposed to a given dose radiation are
fixed, protein lesion-yields are highly variable and closely related to survival.
The Model
Protein Damage
Haber-Weiss
Reaction
complexes
But, the nature of the radioprotective agents and their
targets remained a mystery for 40 years!
Something in D. radiodurans protein-free ultrafiltrates protects proteins
Mn2+
In vitro
BamHI
BamHI
desiccation g-radiation
g-radiation
EC proteins + Ultraftr.
+
!
+
!
!
+
Approach to Isolating Protective Mn2+ Complexes
in Extremely Radiation Resistant Organisms
Protein-Free Extracts
Ultrafiltrates
Ultracentrifugation
+ Ultrafiltration
Small-Molecule Analysis
HPLC
MS - Metabolomics
Chromatography
Atomic Abs Spec
Reconstituted Complexes
Applications
Radioprotection
Enzyme storage
Vaccine preparation
Enzyme radiation activity
assays. Carbonyl assays.
Radioprotection of human cells
and bacteria.
In vitro and in vivo
screening
What is enriched?
Mn(II)
Orthophosphate
Peptides
Nucleosides
Composition of the DR-ultrafiltrate
Mn complexes
d
Free amino acids and peptide-derived amino acids
Putative D. radiodurans Mn2+ Complexes
Orthophosphate
Nucleosideanalogs
O2·-
O2·- HO·
Amino acids/
Peptides
Using various forms of paramagnetic spectroscopy:
Professor Brian Hoffman
Department of Chemistry
Northwestern University
2145 Sheridan Road
Evanston, IL 60208-3113
Ajay Sharma, Elena K. Gaidamakova, Vera Y. Matrosova, Brian Bennett, Michael J. Daly, Brian M.
Hoffman. Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to γ-radiation
by advanced paramagnetic resonance methods. Proc. Natl. Acad. Sci. USA March, 2013
In cells of D. radiodurans, Mn2+:
E.coli
(Sensitive)
● is bound to orthophosphate not polyphosphate
● is bound to water (+)
● mainly bound to small molecules (SM) not enzymes
● coordinated with N (but probably also C=O)
● coordination is not altered by megadoses of g-rays
S.cerevisiae
(Moderately resistant)
+++
+++
+++
++
enzymes
SM
---
---
altered!
altered a bit
Extraordinary Antioxidant Synergism (BamHI Assay)
For example:
25 mM PiB
25 mM
3 mM U
+ BamHI
+ BamHI
25 mM
25 mM PiB 25 mM PiB
3 mM U
3 mM U
1mM MnCl2 1mM MnCl2 1mM MnCl2
+ BamHI
+ BamHI
+ BamHI
+
Then,
BamHI
+
l
Mg2+, Ca2+, Zn2+, Ni2+, Cu2+ and Fe2+ have no protective effect when combined
with uridine (U) and PiB (phosphate buffer, pH 7.4)
Glutamine Synthetase
!!!
We were Stunned!
Aqueous Glutamine Synthetase (GS) Mixture:
D20 GS activity/
g-ray dose:
25 mM PPB (pH7.4):
150 Gy
25 mM PPB (pH7.4)
+ 1 mM Mn:
1,800 Gy
25 mM PPB (pH7.4)
+ 1 mM Mn + 10 mM Leu:
>15,000 Gy
25 mM PPB (pH7.4)
+ 1 mM Mn + 3 mM (A+U):
>25,000 Gy
25 mM PPB (pH7.4)
+ 1 mM Mn + 3 mM Decapeptide:
>40,000 Gy
New Designer “Deinococcus Peptides (NIH) + Mn +Pi
>100,000 Gy!!
Reminder: Survival Curves and DSBs
100%
Survival
Representative
Insects
Rotifers
C. elegans
Fungi
Halobacteria
Cyanobacteria
Deinococci
Amoebae
You and
most
of Life
10%
g-rays
UVC
Desiccation
0
1
0
0
72 DSBs
<1 DSB
5 DSBs 400 DSBs
120 DSBs
10
100
1000
10000
0.1
1
10
100
1000
0.1
1
10
100
1000
Dose/Gy
Dose/J/m2
Dose/days
From Worms to Bacteria: DSB Repair Efficiencies Depend on Protein
Protection
Application
Irradiated Vaccine
All USUHS
Lambda Phage
(See next slide for NIH study)
+ DR Mn-Complexes (Mn-pep-Pi)
tail
tail
DNA is destroyed
40,000 Gy
+ Mn-pep-Pi
40,000 Gy
Proteins survive
Everything is
wiped out
Immunogenic!
DNA
Destroyed
Protein &
Structure
Survives!
Next Slide – Details of Collaboration between Daly and Datta Groups
MRSA
MRSA
VEE VIRUSES
Ex Vivo Radioprotective Effects of Reconstituted
D. radiodurans Mn-Peptide Complexes on Human Cells
100%
Cell Viability %
Reconstituted Complex
DR-ultrafiltrate
Control
0%
100
0
Radiation Dose (Gy)
Collaboration between Daly and Tom Lamkin’s Group
Mn-Complexes applied to the growth medium of E. coli endows
it with extreme radiation resistance under high-level chronic gamma irradiation
The “concentric ring-images” on the agar plates below were developed under
high-level chronic Cs-137 radiation using E. coli growth to show where the
radioprotective Mn complexes were applied.
No Radiation
E. coli
+ 42 Gy/hour
D. radiodurans
D. radiodurans
E. coli
+ 42 Gy/hour
+ 42 Gy/hour
Take-Home Messages:
● At least in prokaryotes, protein oxidation in irradiated cells is not the
consequence of cell death, but its major probable cause – If you want to
survive radiation and other forms of oxidative stress, protect your proteins!
● A direct route to extreme radiation resistance appears to be by metabolic
regulation, ie, via metabolite accumulation, which protects proteins from ROS.
● The possibility that Mn-dependent chemical antioxidants in D. radiodurans are
based on common metabolites raises the possibility that equivalent synergistic
processes promoted by Mn 2+ may be acting similarly in other organisms, and
perhaps also in mitochondria and their mammalian hosts.
● Practical areas which are impacted: 1. bioremediation of high-level radioactive
waste sites; 2. long-duration enzyme/antibody storage; 3. metabolic
interventions at the cellular level which mitigate oxidative stress during
irradiation or aging; and 4. vaccine preparation.
Latest information:
: Daly + Deinococcus
Daly et al., Science 306, 925-1084
(2004)
Daly et al., PLoS Biology, 5(4)
(2007)
Daly et al., ISME J, 2, 393-403
(2008)
Daly, Nat. Rev. Microbiol., 7, 237-45 (2009)
Daly et al., PLoS One., e2349j
(2010)
Daly, DNA Repair 11, 12-21
(2011)
Gaidamakova et al., Cell H-M, 12,
(2012)
Daly and Culotta, Antioxidant&Redox Sig (2012)
Hoffman and Daly et al. PNAS March (2013)
Big Thanks to AFOSR & DTRA et al for Funding
www.youtube.com
Deinococcus radiodurans
Deinococcus: A direct challenge to evolutionary theory
"Creation Moments" daily 2 minute radio/broadcast with host Ian Taylor
is heard around the world on over 1300 stations and outlets.
Each program features scientific evidences of nature that point to delicate
design not evolutionary chance. Creation Moments http://www.creationmoments.net/
“Another wonder of God's design that will not make it into middle or high school textbooks.
Like a lot of other facts, Deinococcus just doesn't fit with the Religion of Evolutionism..”
Research Slide # 5
Bacillus Spores Contain Enzyme-Protecting Mn & Ca Complexes
(DPA)
kGy
Mn2+
kGy
Dipicolinic acid
Putative Structures:
20%
Ca2+
kGy
Mn2+ Ca2+
For comparison,
Blue structure is uridine
The Really Big Question: Death by Protein Damage
in Mammalian Cells?
For many oxidative stress conditions, DNA is no longer considered the principal
target of ROS (radiation, bleach, H2O2, Fe2+, Cu2+, etc) in prokaryotes that
accounts for their toxicity. These trends parallel some of those beginning to
emerge for mammalian cells. For example,
● The relationship between DNA damage and g-ray dose in human cells is about
the same as in all other cell-types (0.005 DSBs/Gy/Genome).
● In cultured mouse cells exposed to g-rays, protein oxidation precedes DNA
damage, and is implicated as a critical and very early event in radiotoxicity.
The new paradigm of radiation toxicity may apply to humans: The key to surviving
radiation: Protect your Proteins! And consider using Deinococcus Mn complexes!
M. J. Daly and K. W. Minton (1995) Resistance to radiation. Science 270, 1318
M. J. Daly (2010) Revising the molecular basis for
radiation effects on cells. Horikoshi, K. (Ed.).
Springer, Japan:
Wilhelm Röntgen (1845–1923)
1901 Nobel Prize in Physics
“On November 8, 1895 Wilhelm C. Roentgen was
studying the passage of an electric current through
a vacuum tube at the Physical Institute of the
University of Wurzburg, Germany. He noticed
that, if the Crookes tube was wrapped in black
cardboard in a dark room, a barium platinocyanide
screen located a few feet away glowed softly.
Because the nature of the invisible light emanating
from the Crookes tube was then unknown,
Roentgen gave them the name X-rays. Within
weeks, his discovery was an international news
story; within months, Roentgen’s original
experiment was being treated as a novelty.
Thomas A. Edison arranged a special exhibit on
Roentgen rays at the annual Electrical Exhibition in
New York City’s Grand Central Palace in May 1896.
This exhibit was a public sensation, mainly due to
his demonstrating on a fluorescent screen the
shadows of the bones of the hands of visitors.
The early success and acceptance in the practical
use of the X-ray in medicine was facilitated by such
public displays. Unfortunately, the dangers of Xrays were not recognized until too late”.
Glass beaker + 15,000 Gy
D. radiodurans survives 15,000 Gy
Control