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?

OH*

Flow Cytometryand HPF

OH*

Argon

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

Conclusion: Fe in Fenton reaction from intracellular source (Fe-S)

Fig 2(again)

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 5: RecA mutant

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!

Final Comments?