A Common Mechanism of Cellular
Death Induced by Bactericidal
Antibiotics
Michael A. Kohanski, Daniel J. Dwyer, Boris Hayete,
Carolyn A. Lawrence, and James J. Collins
Boston University, Boston, MA
Cell. 2007 Sept 7;130( 5): 797-810
Two Anti-Microbial Therapies
• Bactericidal
– Kill >99.9% bacteria
– Specific Drug-Target interactions
• Bacteriostatic
– Inhibit growth of bacteria
• Sometimes not a clear line between two
classes. High concentrations of
bacteriostatic can result in killing.
Classes of Drug-Target
interactions (Bactericidal)
• Inhibit DNA replication/repair
– Quinolines (NORFLOXACIN)
• Inhibit Protein synthesis
– Aminoglycosides (KANAMYCIN)
• Inhibit Cell-wall turnover
– β-lactams (AMPICILLIN)
Question: What is the bacteria’s response to
this primary drug-target interaction?
Bacteriostatic
• Target 30S ribosome function
– Ex: TETRACYCLINE, SPECTINOMYCIN
• Target 50S ribosome function
– Ex: CHLORAMPHENICOL, ERYTHROMYCIN
• Target RNA polymerase
– Ex: RIFAMYCIN
Fenton Reaction
H2O2 + Fe2+
Fe3+ + OH* + OH-
Question: What is the source of Fe?
Ex: Intracellular- Fe-S clusters
Extracellular- Iron Import
Previously, Dwyer et al. showed that gyrase inhibitors
(quinoline synthetic antibiotics) induce breakdown in Fe
regulation, which leads to OH* formation
Question:
*
Does OH formation contribute
to antibiotic-induced cell death
and are there differences
among classes of antibiotics?
Flow Cytometry
and HPF
OH*
OH*
Argon
FIGURE 1: E. coli
Cell Death by H2O2
Hours (post-treatment)
HPF Fluorescence
Log Fluor Intensity
Thiourea: OH* scavenger (sequesters OH*)
Dipyridyl: Iron Chelator (sequesters unbound Fe)
Conclusion: HPF good at measuring hydroxyl radical formation
Amp addition
Cell Death by Bactericidals
FIGURE 1: E. coli
HPF Fluorescence
Note:
Bimodal curve
Amp treated
Conclusion: Bactericidal antibiotics promote formation of OH*
Similar results with
Gram+ Staphylococcus
aureus
Seems killing of cells
correlates to OH*
production
What about lethal doses
of bacteriostatic drugs?
Is OH* formation important in bactericidal killing?
Figure 2
My question:
H2O2 + thiourea = NO OH* as seen by HPF
Norfloxacin + thiourea = some OH* production...
Why the difference?
Fenton Reaction
H2O2 + Fe2+
Fe3+ + OH* + OH-
Where is this iron coming from?
Iron transport/import? TonB mutant
Structure of TonB in Complex with FhuA, E. coli Outer
Membrane Receptor
P.D. Pawelek1, N. Croteau1, C. Ng-Thow-Hing1, C.M.
Khursigara1, N. Moiseeva2, M. Allaire2 and J.W. Coulton1
1Department of Microbiology and Immunology, McGill
University, 2National Synchrotron Light Source, Brookhaven
National Laboratory
tonB no protective effect from bactericidals
Conclusion: Fe in Fenton reaction not from extracellular source
Fenton Reaction
H2O2 + Fe2+
Fe3+ + OH* + OH-
Where is this iron coming from?
Fe-S clusters?
IscS mutant
Lauhon, C. T. et al. J. Biol. Chem. 2004;279:19551-19558
Fig 2
(again)
Conclusion: Fe in Fenton reaction from intracellular source (Fe-S)
Table 1: aka the largest table EVER
Microarray data of genes regulated by bactericidal
drug classes relative to spectinomycin
• Upregulated (38)
– NADH Dehydrogenase ONLY pathway
upregulated by all three!
• Downregulated (142)
– β-lactam resistance membrane protein
– Multidrug efflux system transporter
– Transport, drug/analog sensitivity
• Why spectinomycin?
Fe leaching via superoxides
And how do we get superoxides?
Oxidative Phosphorylation
Complex I
Complex III
H+
Complex IV
ATPase
H+
H+
H+
cyt C
Q
nuo
NUO
ee--
H+
H+
NADH NAD+
NADH
NAD+
Cyt
bc1
cyt
bc1
cyt
aa3
cbb3
1/2 O2
O2
H+
H2O
O2•
ADP
+ Pi
ATP
H+
Figure 3A: Nad+ Cycling Assay
Collect culture, centrifuge, flash freeze pellet
Add NaOH-NADH extraction, HCl-NAD+ extraction (stability)
Boil 10 min, centrifuge, collect supernatant = sample
96 well plate: sample, bicine, neutralizing buffer, phenazine ethosulfate (ecarrier), MTT (redox indicator), EtOH (dissolves MTT), EDTA
Add yeast alcohol dehydrogenase to start reaction-kinetics 570nm
Rate of MTT reduction is
proportional to conc of
NAD+ or NADH
NAD+, NADH standards for
calibration
Figure 3:
Decreased NADH pool
leads to decreased O2
leads to less Fe leaching
leads to less OH* leads to
less death
Repressor
SOS
SOS RESPONSE GENES
DNA Damage
Activates RecA
Promotes LexA
auto cleavage
SOS RESPONSE GENES
AMP
GF
P
Measure Fluorescence
pZ vector
O
ri
Figure 4
Note:
Norfloxacin induces SOS
Bimodal Amp curve
-Corresponds with lysis
No Kan difference
-Requires transcription
AND translation of GFP
-Kan disrupts translation
Figure 5: RecA mutant
Bactericidal Drug
Target not important
Upregulation of NADH
Dehydrogenase
Rapid depletion of NADH
O2*
Destabilize Fe from
Fe-S clusters
Figure 6:
Big Picture
FENTON REACTION!
Hydroxyl Radicals!
DEATH!
Final Comments?