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
A Common Mechanism of CellularDeath 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 twoclasses. High concentrations ofbacteriostatic can result in killing.
Classes of Drug-Targetinteractions (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 tothis 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 ReactionH2O2 + Fe2+ Fe3+ + OH* + OH-
Previously, Dwyer et al. showed that gyrase inhibitors(quinoline synthetic antibiotics) induce breakdown in Fe
regulation, which leads to OH* formation
Question: What is the source of Fe?Ex: Intracellular- Fe-S clusters
Extracellular- Iron Import
Question:Does OH* formation contributeto antibiotic-induced cell death
and are there differencesamong classes of antibiotics?
Thiourea: OH* scavenger (sequesters OH*)Dipyridyl: Iron Chelator (sequesters unbound Fe)
Cell Death by H2O2 HPF Fluorescence
Hours (post-treatment) Log Fluor Intensity
Conclusion: HPF good at measuring hydroxyl radical formation
FIGURE 1: E. coli
Cell Death by Bactericidals HPF Fluorescence
Note:Bimodal curveAmp treated
FIGURE 1: E. coli
Conclusion: Bactericidal antibiotics promote formation of OH*
Amp addition
Similar results withGram+ Staphylococcus
aureus
Seems killing of cellscorrelates to OH*
production
What about lethal dosesof 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 ReactionH2O2 + Fe2+ Fe3+ + OH* + OH-
Where is this iron coming from?
Iron transport/import? TonB mutant
Structure of TonB in Complex with FhuA, E. coli OuterMembrane 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, McGillUniversity, 2National Synchrotron Light Source, Brookhaven
National Laboratory
tonB no protective effect from bactericidals
Conclusion: Fe in Fenton reaction not from extracellular source
Fenton ReactionH2O2 + 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
Table 1: aka the largest table EVERMicroarray 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 superoxidesAnd how do we get superoxides?
Oxidative Phosphorylation
Complex I Complex III Complex IV ATPase
nuo
H+
H+ H+
H+H+
H+
H+
H+
ADP + Pi
ATPH2O1/2 O2
NADH NAD+
e-
cyt C
cyt bc1
cyt aa3cbb3Q Cyt
bc1e-
NUO
NADH NAD+O2
O2•
Figure 3A: Nad+ Cycling AssayCollect 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 (e-carrier), 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 NADHNAD+, NADH standards for
calibration
Decreased NADH poolleads to decreased O2
leads to less Fe leachingleads to less OH* leads to
less death
Figure 3:
SOS RESPONSE GENES
SOS RESPONSE GENES
Repressor
DNA DamageActivates RecAPromotes LexAauto cleavage
SOS
GFP
pZ vectorAM
P
Ori
Measure Fluorescence
Figure 4
Note:
Norfloxacin induces SOS
Bimodal Amp curve-Corresponds with lysis
No Kan difference
-Requires transcriptionAND translation of GFP
-Kan disrupts translation
Figure 6:Big Picture
Bactericidal DrugTarget not important
Upregulation of NADHDehydrogenase
Destabilize Fe fromFe-S clusters
Rapid depletion of NADH O2*
FENTON REACTION!
Hydroxyl Radicals!DEATH!